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WO2007074010A1 - Procede de production de l’acide trans-10, cis 12 octadecadienoique - Google Patents

Procede de production de l’acide trans-10, cis 12 octadecadienoique Download PDF

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Publication number
WO2007074010A1
WO2007074010A1 PCT/EP2006/069030 EP2006069030W WO2007074010A1 WO 2007074010 A1 WO2007074010 A1 WO 2007074010A1 EP 2006069030 W EP2006069030 W EP 2006069030W WO 2007074010 A1 WO2007074010 A1 WO 2007074010A1
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Prior art keywords
linoleic acid
cla
cis
trans
conjugated linoleic
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Catherine Stanton
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Teagasc Dairy Products Research Centre
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Teagasc Dairy Products Research Centre
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Priority to EP06830179A priority Critical patent/EP1963517A1/fr
Priority to US12/158,808 priority patent/US20090105341A1/en
Priority to JP2008546335A priority patent/JP2009521213A/ja
Publication of WO2007074010A1 publication Critical patent/WO2007074010A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
    • A23L33/12—Fatty acids or derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C3/00—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom
    • C11C3/14—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom by isomerisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
    • C12P7/6431—Linoleic acids [18:2[n-6]]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6472—Glycerides containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the present invention also relates to feed-, food-products and nutraceuticals enriched in conjugated linoleic acid and to transgenic microorganisms expressing an alien gene encoding a trans-10, cis-12 conjugated linoleic acid isomerase and to the use of the same as probiotics in food or feed.
  • An additional embodiment of the current invention relates to the fermented oil produced according to the inventive method and the use of said fermented oil for the production of medicaments.
  • Fatty acids and triglycerides have a multiplicity of applications in the food industry, animal nutrition, cosmetics and in the pharmaceutical sector. Depending on whether they are free saturated or unsaturated fatty acids or triglycerides with an increased content of saturated or unsaturated fatty acids, they are suitable for a very wide range of applications; thus, for example, polyunsaturated fatty acids are added to baby formula to increase the nutritional value.
  • the various fatty acids and triglycerides are obtained mainly from microorganisms such as Mortierella or from oil-producing plants such as soya, oilseed rape, sunflowers and others, where they are usually obtained in the form of their triacyl glycerides. Alternatively, they are obtained advantageously from animals, such as fish.
  • the free fatty acids are prepared advantageously by hydrolysis.
  • oils with unsaturated or with saturated fatty acids are preferred depends on the intended purpose; thus, for example, lipids with unsaturated fatty acids, specifically polyunsaturated fatty acids, are preferred in human nutrition since they have a positive effect on the cholesterol level in the blood and thus on the possibility of heart disease. They are used in a variety of dietetic foodstuffs or medicaments.
  • conjugated unsaturated fatty acids are the so-called conjugated unsaturated fatty acids, such as conjugated linoleic acid.
  • conjugated fatty acids such as conjugated linoleic acid.
  • a series of positive effects have been found for conjugated fatty acids; thus, the administration of conjugated linoleic acid reduces body fat in humans and animals, and increases the conversion of feed into body weight in the case of animals (WO 94/16690, WO 96/06605, WO 97/46230, WO 97/46118).
  • conjugated linoleic acid it is also possible to positively affect, for example, allergies (WO 97/32008) or cancer (Banni et al., Carcinogenesis, Vol. 20, 1999: 1019- 1024, Thompson et al., Cancer, Res., Vol. 57, 1997: 5067- 5072).
  • t10, c12 CLA isomer specifically down regulates triglyceride accumulation and PPARgamma expression in human pre-adipocytes as well as mature adipocytes.
  • Human CLA supplementation to a group of 53 healthy men and women (4.2 g/d; equal amounts c9, t11 and t10, c12 CLA) reduced the proportion of body fat by 3.8 % compared with the control group given olive oil (Smedman and Vessby, 2001).
  • CLA plays an important role in health promotion and that specifically the t10, c12 CLA isomer may be useful in treatment of overweight and obese animal and human subjects. By enrichment of this isomer and incorporation into functional foods and thus make it available on a daily basis, it may have a large potential in prevention and treatment of these conditions.
  • Both the t10, c12 and the c9, t11 CLA isomers have been reported to exert anti-carcinogenic activity. In particular, it has been shown to inhibit skin tumor initiation and forestomach neoplasia as well as inhibiting chemically induced skin tumor promotion and mammary and colon tumorigenesis (Belury, 2002).
  • the mechanisms by which CLA exerts the many physiological effects is not yet fully understood, but at least two different models have been proposed.
  • One model suggests that CLA reduces the arachidonate pool leading to a reduced production of downstream eicosanoid products, which modulates cytokine production involved in inflammation and cancer.
  • the other model includes regulation of expression of genes known to control lipid oxidation, adipocyte differentiation, energy balance and atherogenesis (Beluri, 2002; Pariza et al, 2000).
  • CLA can be manufactured synthetically from alkaline isomerization of linoleic and linolenic acids, or vegetable oils containing linoleic or linolenic acids. Two reactions are catalyzed when heating oil at 180 0 C under alkaline conditions; hydrolysis of the fatty acid ester bond from the triglyceride lipid backbone, which produces free fatty acids, and conjugation of unconjugated unsaturated fatty acids with two or more appropriate double bonds (WO 99/32604). This method produces about 20-35% cis-9, trans-11 CLA and about the same amount of trans-10, cis-12 CLA, but enrichment of either of the isomers relative to the other is possible by using a fractional crystallization procedure. In addition, other isomers are produced mainly trans, trans isomers.
  • conjugated fatty acids for example conjugated linoleic acid
  • US 3,356,699 and US 4,164,505 The chemical preparation of conjugated fatty acids, for example conjugated linoleic acid, is also described in US 3,356,699 and US 4,164,505.
  • CLA is formed naturally as an intermediate during biohydrogenation of linoleic acid by rumen bacteria, and natural sources of CLA are consequently milk and fats from ruminants.
  • the main CLA isomer in milk fat is the c9, t11 CLA, which accounts for 80-90 % of total milk fat CLA, whereas the t10, c12 CLA isomer is only present at about 1 % (Jensen, 2002).
  • a range of cultures with ability to convert linoleic acid into the c9, t11 CLA isomer are known, in addition to the rumen microflora.
  • WO 99/29886 describes the use of bacterial strains found among food grade bacteria, particularly among dairy starter cultures, which have the ability to produce CLA in vitro by fermentation.
  • WO 99/32604 describes a linoleate isomerase from Lactobacillus reuteri.
  • the enzyme activity leads to the conversion of linoleic acid to six different CLA species which are as follows: (cis,trans)-9,11-CLA, (trans,cis)-10,12-CLA, (cis,cis)-9,11-CLA, (cis,cis)-10,12-CLA, (trans,trans)-9,11-CLA and (trans,trans)-10,12-CLA.
  • the disadvantages of using the above-mentioned isomease is that the yield of the reaction is very low, the purity of the CLA produced is for an industrial process not sufficient and the process takes place with only low space-time yields. This leads to economically unattractive processes.
  • transgenic micororganisms belonging to the family of Lactobacillaceae, Streptococcaceae, Propionibacteriaceae, Enterobacteriaceae or Bifidobacteriaceae expressing a nucleic acid molecule encoding a CLA isomerase, particularly a trans-10, cis-12 conjugated linoleic acid isomerase.
  • trans-10, cis-12 conjugated linoleic acid isomerase enabled the transgenic organisms to convert 50% of the added linoleic acid into trans-10, cis-12 conjugated linoleic acid when expressed in Lactococcus lactis, followed by 40% and 30% conversion rates by E.coli and Lactobacillus paracasei, respectively.
  • a first subject matter of the invention therefore relates to a process for the production of trans- 10, cis-12 conjugated linoleic acid in a transgenic microorganism comprising the steps of: (a) introducing into said microorganism at least one nucleic acid molecule encoding a trans- 10, cis-12 conjugated linoleic acid isomerase, (b) culturing the transgenic microorganism obtained under (a),
  • trans-10, cis-12 conjugated linoleic acid isomerase is characterized by a sequence i. as described by SEQ ID No. 1 , or ii. having at least 50 consecutive base pairs of the sequence described by SEQ ID No.1 , or iii. having an identity of at least 80% over a sequence of at least 100 consecutive nucleic acid base pairs to the sequence described by SEQ ID No. 1 , or iv. hybridizing under high stringent conditions with a nucleic acid fragment of at least 50 consecutive base pairs of a nucleic acid molecule described by SEQ ID No.1 , or v. encoding a polypeptide having at least 75% identity to the amino acid sequence as shown in SEQ ID No. 2 and encoding a trans-10, cis-12 conjugated linoleic acid isomerase.
  • trans-10, cis-12 conjugated linoleic acid isomerase is isolated from a rumen bacteria, preferably from Megashera elsdenii YJ-4.
  • the invention relates to the above described process, wherein the nucleic acid molecule encoding said trans-10, cis-12 conjugated linoleic acid isomerase is isolated from a microorganism belonging to the genus Propionibacterium, preferably from Propionibacterium acnes.
  • the invention relates to a process for the production of conjugated linoleic acid in a transgenic microorganism according to the above described steps (a) to (e), characterized in that the microorganism used under (a) belong to the family selected from the group consiting of Lactobacillaceae, Streptococcaceae, Propionibacteriaceae, Enterobacteriaceae and Bifidobacteriaceae, preferably the used microorganism belong to the genus selected from the group consiting of Lactococcus, Lactobacillus, Propionibacterium,
  • Escherichia and Bifidobacterium more preferably said microorganism is selected from group consisting of Lactococcus lactis, Lactobacillus paracasei and Escherichia coli.
  • the process for the production of conjugated linoleic acid in a transgenic microorganism is characterized in that the linoleic acid is added to a microorganism culture having an optical density (OD ⁇ oo) of at least 0.1.
  • the invention relates to a process for the production of conjugated linoleic acid in a transgenic microorganism according to the above described steps (a) to (e), characterized in that the bioconversion rate of linoleic acid is higher than 10%.
  • the invention relates to a process for the production of feed or food products or nutraceuticals enriched in conjugated linoleic acid, wherein the used conjugated linoleic acid is produced according to the above described process.
  • the invention relates furthermore to feed-, food-products and nutraceuticals enriched in conjugated linoleic acid, wherein the conjugated linoleic acid is produced according to the above described process.
  • the invention relates to transgenic microorganisms expressing a nucleic acid molecule encoding a trans-10, cis-12 conjugated linoleic acid isomerase characterized by a sequence (i) as described by SEQ ID No. 1 , or (ii) having at least 50 consecutive base pairs of the sequence described by SEQ ID No.1 , or (iii) having an identity of at least 80% over a sequence of at least 100 consecutive nucleic acid base pairs to the sequence described by SEQ ID No. 1 , or (iv) hybridizing under high stringent conditions with a nucleic acid fragment of at least 50 consecutive base pairs of a nucleic acid molecule described by SEQ ID No.
  • nucleic acid sequence is preferably isolated from a rumen bacteria, more preferably from Megashera elsdenii, most preferably from Megashera elsdenii YJ-4, or from a microorganism belonging to the genus Propionibacterium, preferably Propionibacterium acnes , wherein said nucleic acid molecule is functionally linked to at least one heterologous promoter sequence.
  • the present invention relates to the use of the inventive transgenic microorganism, preferably microorganism belonging to the genus selected from the group consisting of Lactococcus, Lactobacillus, Propionibacterium, Escherichia and Bifidobacterium, more preferably microorganism selected from the group consisting of Bifidobacterium breve, Bifidobacterium dentium and Bifidobacterium pseudocatenulatum as probiotics in food and feed.
  • the inventive transgenic microorganism preferably microorganism belonging to the genus selected from the group consisting of Lactococcus, Lactobacillus, Propionibacterium, Escherichia and Bifidobacterium, more preferably microorganism selected from the group consisting of Bifidobacterium breve, Bifidobacterium dentium and Bifidobacterium pseudocatenulatum as probiotics in food and feed.
  • the invention relates to fermented oil produced in a transgenic microorganism according to the above described inventive process.
  • the invention relates furthermore to the use of the fermented oil produced according to the above described inventive method for the production of a medicament for the treatment of cancer.
  • Fig. 1 The pNZ44-coPAI construct (SEQ ID No. 5).
  • Fig. 4 CLA production vs linoleic acid (LA) usage and accumulation of the fatty acids in the membranes by E. coli pNZ44-coPAI incubated with 0.5 mg/ml LA for 72 hours.
  • LA linoleic acid
  • Fig. 5 Cell viability for SW480 cells treated with 5-25 ⁇ g fermented oils/fatty acids/ml media after 5 days incubation. Data represents cell viability expressed as percentage of ethanol control, which was set to be 100%.
  • A GLC profile of LA control oil extracted from LB media following 72 hours incubation in 37 0 C and cell viability
  • B of SW480 following treatment with LA control oil.
  • C GLC profile of GM17 media following 72 hour growth of
  • L. lactis pNZ44-coPAI in 0.5 mg/ml LA and cell viability D) following treatment with L. lactis t10, c12 CLA (fermented oil).
  • E GLC profile of LB media following growth of E. coli pNZ44-coPAI in 0.5 mg/ml LA and cell viability (F) following treatment with E. coli t10, c12 CLA (fermented oil).
  • G Cell viability following treatment with the pure synthetic t10, c12 CLA isomer (Matreya) and (H) linoleic acid (Sigma).
  • Fig. 6 Microscopic examination of human colon cancer cells SW480 following 5 days incubation with different oils/fatty acids.
  • A Linoleic acid unfermented control oil, 5 ⁇ g/ml media (100 X magnification) and (B) 25 ⁇ g/ml media (200 x).
  • C E. coli t10, c12 CLA (fermented oil), 5 ⁇ g/ml media (100 X) and (D) 20 ⁇ g/ml media (200 X).
  • E L. lactis t10, c12 CLA (fermented oil), 5 ⁇ g/ml media (100 X) and (F) 20 ⁇ g/ml media (200 X).
  • Animal refers to an organism taxonomically assigned to the animal kingdom (animalia). Those which are preferred are the vertebrates (vertebrata) with the orders of the tetrapoda (land vertebrates) and fish (pisces). Particular preference is given to the classes aves (birds) and mammalia (mammals), modern humans (Homo sapiens) being comprised as a particularly preferred mammal.
  • bioconversion rate as used herein in reference to the production of conjugated linoleic acid, preferably trans-10, cis-12 conjugated linoleic acid refers to the amount of free linoleic acid given in percent that has been converted to conjugated linoleic acid after a certain fermentation period or at the end of the fermentation process.
  • cis-12 conjugated linoleic acid isomerase 0.5 mg/ml linoleic acid was added and incubation continued for 72 hours, followed by extraction of fatty acids from samples taken from the broth. The ratio of CLA/LA in the samples is determined using GLC
  • Cell refers to a single cell.
  • the term "cells" refer to a population of cells.
  • the population may be a pure population comprising one cell type. Likewise, the population may comprise more than one cell type. In the present invention, there is no limit on the number of cell types that a cell population may comprise.
  • the cells may be synchronized or not synchronized, preferably the cells are synchronized.
  • Coding region or coding sequence when used in reference to a gene refers to the nucleotide sequences which encode the amino acids found in the nascent polypeptide as a result of translation of a mRNA molecule.
  • the coding region is bounded, in eucaryotes, on the 5' -side by the nucleotide triplet "ATG" which encodes the initiator methionine and on the 3'-side by one of the three triplets, which specify stop codons (i.e., TAA, TAG, TGA).
  • Conjugated linoleic acid refers to a mixture of positional and geometric isomers of linoleic acid, involving double bonds at positions 7 and 9, 9 and 11 , 10 and 12 or 11 and 13.
  • the isomers cis-9, trans-11 and trans-10, cis-12 isomers are of particular interest, because many beneficial effects have been attributed to said isomers.
  • the isomers can differ positionally (mainly at positions 7 and 9, 9 and 11 , or 10 and 12) (Ha et al., Anticarcinogens from fried ground beef: heat-altered derivatives of linoleic acid. Carcinogenesis.
  • This isomer is also the predominant dietary form of CLA, obtained from fats derived from ruminant animals, including milk, dairy products and meat (Chin et al., Dietary sources of conjugated dienoic isomeres of linoleic acid, a newly recognized class of anticarcinogens. J. Food Comp. and Anal.5: 185-197 (1992)).
  • the terms trans-10, cis-12 octadecadienoic acid and trans-10, cis-12 CLA are used herein interchangeably.
  • Conjugated linoleic acid isomerase is a protein catalizing the isomerization of linoleic acid or conjugated linoleic acid isomers, characterized in that a double bond at one carbon position is transferred to another carbon position forming one of the possible CLA isomers.
  • Trans-10, cis-12 conjugated linoleic acid isomerase as used in the context of this invention means an enzyme catalysing the isomerisation of linoleic acid to trans-10, cis-12 octadecadienoic acid.
  • trans-10, cis-12 octadecadienoic acid isomerase and trans-10, cis-12 CLA isomerase are used herein interchangeably.
  • microorganism in liquid culture under controlled conditions. Depending on the organisms used in the processes the growth conditions can be very different and are in general known to those skilled in the art.
  • microorganism are grown in a liquid medium which contains a carbon source, usually in the form of sugars, a nitrogen source, usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, a phosphate source such as potassium hydrogen phosphate, trace elements such as iron salts, manganese salts, magnesium salts and, if required, vitamins, at temperatures between 0 0 C and 100 0 C, preferably between 10 0 C and 65°C, 15°C and 55°C, more preferably between 20 0 C and 50 0 C, 25°C and 45°C, particularly preferred between 30 0 C and 40 0 C while gassing in oxygen.
  • a carbon source usually in the form of sugars
  • a nitrogen source usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium s
  • the organism can be grown under aerobic or anaerobic conditions.
  • the pH of the liquid medium can be maintained at a fixed value, i.e. the pH is regulated while culture takes place.
  • the pH should then be in a range between pH 2 and pH 9, preferably between 4 and 8.5, 4.5 and 8, more preferably between 5 and 7.5, 5.5 and 7.
  • the microorganisms may also be cultured without pH regulation.
  • Culturing can be effected by the batch method, the semi-batch method or continuously Nutrients may be supplied at the beginning of the fermentation or fed in semicontinuously or continuously.
  • Such methods can be found in e.g Scardovi V (1986) Genus Bifidobacterium and Genus Lactobacillus. In Bergey's Manual of Systematic Bacteriology [NM PHA Sneath, ME Sharpe, JG Holt, editor]. Baltimore: Williams & Wilkins.
  • Expression refers to the biosynthesis of a gene product.
  • expression involves transcription of the structural gene into mRNA and - optionally - the subsequent translation of mRNA into one or more polypeptides.
  • fermented oil refers to the oil and fatty acid containing fraction produced by a microorganism during a fermentation. Fermentation is used to refer to the bulk growth of microorganisms on a growth medium. No distinction is made between aerobic and anaerobic metabolism when the word is used in the context of the present invention.
  • fermented oil refers to the fatty acids fraction that can be recovered/isolated (e.g. see example 8) from the microorganism, particularly the cell membranes of the microorganism or the fermentation broth.
  • nucleic acid sequence has to be understood as natural or artificial mutations of the SEQ ID No. 1. Mutations can be insertions, deletions or substitutions of one or more nucleic acids that do not diminish the Linoleic acid isomeration activity of the expression product of said sequence.
  • These functional equivalents having a identity of at least 80%, preferably 85%, more preferably 90%, most preferably more than 95%, very especially preferably at least 98% identity - but less then 100% identity to the sequence as described by the SEQ ID No. 1 , wherein said identity is determined over a sequence of at least 100 consecutive base pairs, preferably at least 150 consecutive base pairs, more preferably at least 200 consecutive base pairs of the sequence as described by any of the SEQ ID No. land having essentially the same enzymatic activity as the sequence shown in SEQ ID No. 2.
  • homologs when used in reference to conjugated linoleic acid isomerases refers orthologs as well as paralogs of the nucleic acid molecule as shown in SEQ ID No.1. These orthologs or paralogs encoding for proteins sharing more than 60%, preferably 65%, 70%, 75%, 80%, more preferably 85%, 90%, 95% or most preferably more than 95% sequence identity on amino acid level with SEQ ID No. 2, wherein said identity is determined over a sequence of at least 100 consecutive amino acids, preferably at least 150 consecutive amino acids, more preferably at least 200 consecutive amino acids of the sequence as described by any of the SEQ ID No. 2 and having essentially the same enzymatic activity as the sequence shown in SEQ ID No. 2.
  • Functional equivalents as described above might have, compared to the trans-10, cis-12 conjugated linoleic acid isomerase from Propionibacterium acnes (SEQ ID No.1) a reduced or increased enzymatic activity or bioconversion rate.
  • the enzymatic activity or bioconversion rate of the functional equivalent is at least 50% higher, preferably at least 100% higher, especially preferably at least 300% higher, very especially preferably at least 500% higher than a reference value obtained with the trans-10, cis-12 conjugated linoleic acid isomerase from Propionibacterium acnes (SEQ ID No.1) under otherwise unchanged conditions.
  • Functionally linked or operably linked is to be understood as meaning, for example, the sequential arrangement of a regulatory element (e.g. a promoter) with a nucleic acid sequence to be expressed and, if appropriate, further regulatory elements (such as e.g., a terminator) in such a way that each of the regulatory elements can fulfill its intended function to allow, modify, facilitate or otherwise influence expression of said nucleic acid sequence.
  • the expression may result depending on the arrangement of the nucleic acid sequences in relation to sense or antisense RNA. To this end, direct linkage in the chemical sense is not necessarily required. Genetic control sequences such as, for example, enhancer sequences, can also exert their function on the target sequence from positions that are further away, or indeed from other DNA molecules.
  • conjugated linoleic acid isomerase refers to the linkage of at least one of isomerase to a nucleic acid sequences in a way that the isomerase can be produced or synthesized in the host cell harbouring said DNA molecule.
  • Expression constructs, wherein the trans-10, cis-12 conjugated linoleic acid isomerase from Propionibacterium acnes (SEQ ID No.1) is functionally linked to an promoter are shown in the examples.
  • Operable linkage, and an expression cassette can be generated by means of customary recombination and cloning techniques as are described, for example, in Maniatis T, Fritsch EF and Sambrook J (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY), in Silhavy TJ, Berman ML and Enquist LW (1984) Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY), in Ausubel FM et al. (1987) Current Protocols in Molecular Biology, Greene Publishing Assoc, and Wiley lnterscience and in Gelvin et al. (1990) In: Plant Molecular Biology Manual.
  • sequences which, for example, act as a linker with specific cleavage sites for restriction enzymes, or as a signal peptide, may also be positioned between the two sequences.
  • the insertion of sequences may also lead to the expression of fusion proteins.
  • the expression construct consisting of a linkage of a promoter and a nucleic acid sequence to be expressed, can exist in a vector-integrated form and be inserted into a bacterial genome, for example by transformation.
  • Gene refers to a coding region operably linked to appropriate regulatory sequences capable of regulating the expression of the polypeptide in some manner.
  • a gene includes untranslated regulatory regions of DNA (e.g., promoters, enhancers, repressors, etc.) preceding (upstream) and following (downstream) the coding region (open reading frame, ORF) as well as, where applicable, intervening sequences (i.e., introns) between individual coding regions (i.e., exons). Genes may also include sequences located on both the 5'- and 3'-end of the sequences, which are present on the RNA transcript.
  • flanking sequences or regions are located 5' or 3' to the non-translated sequences present on the mRNA transcript.
  • the 5'-flanking region may contain regulatory sequences such as promoters and enhancers, which control or influence the transcription of the gene.
  • the 3'- flanking region may contain sequences, which direct the termination of transcription, posttranscriptional cleavage and polyadenylation.
  • Genome and genomic DNA of an organism is the whole hereditary information of an organism that is encoded in the DNA (or, for some viruses, RNA). This includes both the genes and the non-coding sequences.
  • the term "chromosomal DNA” or “chromosomal DNA sequence” is to be understood as the genomic DNA of the cell independent from the cell cycle status. Chromosomal DNA might therefore be organized in different forms, they might be condensed or uncoiled. An insertion into the chromosomal DNA can be demonstrated and analyzed by various methods known in the art like e.g., polymerase chain reaction (PCR) analysis, Southern blot analysis, fluorescence in situ hybridization (FISH), and in situ PCR.
  • PCR polymerase chain reaction
  • FISH fluorescence in situ hybridization
  • Heterologous with respect to a nucleic acid sequence refers to a nucleotide sequence, which is ligated to a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature.
  • Hybridizing includes "any process by which a strand of nucleic acid joins with a complementary strand through base pairing.” (Coombs 1994, Dictionary of Biotechnology, Stockton Press, New York N. Y.). Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, the Tm of the formed hybrid, and the G:C ratio within the nucleic acids. As used herein, the term “Tm” is used in reference to the “melting temperature.” The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands.
  • hybridization conditions may be employed to comprise either low or high stringency conditions; factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of either low or high hybridization stringency.
  • factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of either low or high hybridization stringency.
  • Identity when used in relation to nucleic acids refers to a degree of complementarity. Identity between two nucleic acids is understood as meaning the identity of the nucleic acid sequence over in each case the entire length of the sequence, which is calculated by comparison with the aid of the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA) with the parameters being set as follows: Gap Weight: 12 Length Weight: 4 Average Match: 2,912 Average Mismatch:-2,003
  • a sequence with at least 95% identity to the sequence SEQ ID No. 1 at the nucleic acid level is understood as meaning the sequence that, upon comparison with the sequence SEQ ID No. 1 by the above program algorithm with the above parameter set, has at least 95% identity.
  • inducing when used in relation to the inventive process refers to the inoculation of cell cultures with (i) linoleic acid, or (ii) a expression inducing agent, in the case that the promoter used to drive the expression of a conjugated linoleic acid isomerase is an inducible promoter,
  • introducing refers to a recombinant DNA expression construct that will be introduced into the bacterial cell.
  • the term introducing encompasses for example methods such as transfection, transduction or transformation.
  • Isolating when used in relation to the produced conjugated linoleic acid accordinging to the inventive process refers to the process of extracting (i) the fermentative oil, or (ii) the fatty acid/lipid fraction, or (iii) the conjugated linoleic acid, or (iv) the trans-10, cis-12 conjugated linoleic acid from the fermentation broth, the bacterial pellets/bacterial cell membranes or the supernatant after centrifugation of the fermentation broth (see examples).
  • the isolation can be done from batch-operations or fed-batch-operations.
  • batch-operations all ingredients used in the operation are fed to the processing vessel at the beginning of the operation and no addition or withdrawal of material takes place during the fermentation process.
  • fed-batch operations material can be added or harvested during the fermentation process.
  • Microorganism refers to yeast species and bacteria as defined by Woese (Woese et al., "Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya.” Proc. Natl. Acad. Sci.
  • microorganism that belong to the family selected from the group consiting of Lactobacillaceae, Streptococcaceae, Propionibacteriaceae, Enterobacteriaceae and Bifidobacteriaceae, more preferably the used microorganism belong to the genus selected from the group consiting of Lactococcus, Lactobacillus, Propionibacterium, Escherichia and Bifidobacterium, particularly preferably said microorganism is selected from group consisting of Lactococcus lactis, Lactobacillus paracasei and Escherichia colito microorganism, including Lactobacillus species, Bifidobacterium species, Lactococcus species and yeasts
  • Nucleic acid refers to deoxyribonucleotides, ribonucleotides or polymers or hybrids thereof in single-or double-stranded, sense or antisense form. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.
  • the term “nucleic acid” can be used to describe a "gene”, “cDNA”,”DNA” “mRNA”, “oligonucleotidea”nd “polynucl ⁇ tide”.
  • Nucleic acid sequence refers to the consecutive sequence of deoxyribonucleotides or ribonucleotides (nucleotides) of a DNA fragment (oligonucleotide, polynucleotide, genomic DNA, cDNA etc.) as it can made be available by DNA sequencing techniques as a list of abbreviations, letters, characters or words, which represent nucleotides.
  • Nucleic acid molecule refers to the physically DNA molecule present in the genomic DNA, an appropriate vector or plasmid.
  • the nucleic acid molecule is defined by a nucleic acid sequence.
  • nutraceutical is a combination of "nutritional” and “pharmaceutical” and refers to foods thought to have a beneficial effect on human health.
  • a nutraceutical is any substance that is a food or a part of a food and provides medical or health benefits, including the prevention and treatment of disease. Such products may range from isolated nutrients, dietary supplements and specific diets to genetically engineered designer foods, herbal products, and processed foods such as cereals, soups and beverages
  • Optical density (or absorbance): of a bacterial culture is the turbidity (optical density) of said culture.
  • turbidity optical density
  • a spectrophotometer light passing through a sample is measured by a photoelectric cell. As cell density of the sample increases, i.e., becomes more turbid, a greater amount of light is scattered and fails to reach the photoelectric cell. This is measured in terms of optical density (OD) or absorbance (A) units.
  • I transmitted light; amount of light passing through sample on to the photoelectric cell .
  • a standard curve can be generated that relates cell numbers or mass to optical density readings, i.e., determine both the optical density and cell numbers (or mass) for a series of samples containing different amounts of microorganisms.
  • Probiotics are defined as live microorganisms, including Lactobacillus species, Bifidobacterium species, Lactococcus species and yeasts, that may beneficially affect the host upon ingestion by improving the balance of the intestinal microflora.
  • Bifidobacteria are normal inhabitants of the human and animal colon. Newborns, especially those that are breast-fed, are colonized with bifidobacteria within days after birth. Bifidobacteria were first isolated from the feces of breast-fed infants. The population of these bacteria in the colon appears to be relatively stable until advanced age when it appears to decline. The bifidobacteria population is influenced by a number of factors, including diet, antibiotics and stress. Bifidobacteria are gram-positive anaerobes. They are non-motile, non-spore forming and catalase-negative. They have various shapes, including short, curved rods, club-shaped rods and bifurcated Y-shaped rods.
  • guanine and cytosine content of their DNA is between 54 mol% and 67mol%. They are saccharolytic organisms that produce acetic and lactic acids without generation of CO2, except during degradation of gluconate. They are also classified as lactic acid bacteria (LAB). To date, 30 species of bifidobacteria have been isolated.
  • Bifidobacteria used as probiotics include Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium animalis, Bifidobacterium thermophilum, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis and Bifidobacterium lactis.
  • Specific strains of bifidobacteria used as probiotics include Bifidobacterium breve strain Yakult, Bifidobacterium breve RO7O, Bifidobacterium lactis Bb12, Bifidobacterium longum RO23, Bifidobacterium bifidum RO71 , Bifidobacterium infantis RO33, Bifidobacterium longum BB536 and Bifidobacterium longum SBT-2928.
  • LACTOBACILLUS Lactobacilli are normal inhabitants of the human intestine and vagina. Lactobacilli are gram- positive facultative anaerobes.
  • Lactobacilli are non-spore forming and non-flagellated rod or coccobacilli.
  • the guanine and cytosine content of their DNA is between 32 mol% and 51 mol%. They are either aerotolerant or anaerobic and strictly fermentative. In the homofermentative case, glucose is fermented predominantly to lactic acid. Lactobacilli are also classified as lactic acid bacteria (LAB). To date, 56 species of the genus Lactobacillus have been identified. Lactobacilli used as probiotics include Lactobacillus acidophilus, Lactobacillus brevis,
  • Lactobacillus bulgaricus Lactobacillus casei, Lactobacillus cellobiosus, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus fermentum, Lactobacillus GG (Lactobacillus rhamnosus or Lactobacillus casei subspecies rhamnosus), Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus plantarum and Lactobacillus salivarus. Lactobacillus plantarum 299v strain originates from sour dough. Lactobacillus plantarum itself is of human origin.
  • Lactobacillus acidophilus BG2FO4 Lactobacillus acidophilus INT-9, Lactobacillus plantarum ST31 , Lactobacillus reuteri, Lactobacillus johnsonii LA1 , Lactobacillus acidophilus NCFB 1748, Lactobacillus casei Shirota, Lactobacillus acidophilus NCFM, Lactobacillus acidophilus DDS-1 , Lactobacillus delbrueckii subspecies delbrueckii, Lactobacillus delbrueckii subspecies bulgaricus type 2038, Lactobacillus acidophilus SBT -2062, Lactobacillus brevis, Lactobacillus salivarius UCC 118 and Lactobacillus paracasei subsp paracasei F19.
  • Lactobacillus plantarum ST31 Lactobacillus
  • Lactococci are gram-positive facultative anaerobes. They are also classified as lactic acid bacteria (LAB). Lactococcus lactis (formerly known as Streptococcus lactis) is found in dairy products and is commonly responsible for the souring of milk.
  • Lactococci that are used or are being developed as probiotics include Lactococcus lactis, Lactococcus lactis subspecies cremoris (Streptococcus cremoris), Lactococcus lactis subspecies lactis NCDO 712, Lactococcus lactis subspecies lactis NIAI 527, Lactococcus lactis subspecies lactis NIAI 1061 , Lactococcus lactis subspec ies lactis biovar diacetylactis NIAI 8 W and Lactococcus lactis subspecies lactis biovar diacetylactis ATCC 13675.
  • Promoter refers to a DNA sequence which when ligated to a nucleotide sequence of interest is capable of controlling the transcription of the nucleotide sequence of interest into mRNA.
  • a promoter is a recognition site on a DNA sequence that provide an expression control element for a gene and to which RNA polymerase specifically binds and initiates RNA synthesis (transcription) of that gene.
  • a promoter is typically, though not necessarily, located 5' (i.e., upstream) of a nucleotide sequence of interest (e.g., proximal to the transcriptional start site of a structural gene).
  • constitutive when made in reference to a promoter means that the promoter is capable of directing transcription of an operably linked nucleic acid sequence in the absence of a stimulus (e.g., heat shock, chemicals, light, etc.).
  • constitutive promoters are capable of directing expression of a transgene in substantially any physiological conditions of a cell.
  • a “regulatable” promoter is one which is capable of directing a level of transcription of an operably linked nuclei acid sequence in the presence of a stimulus (e.g., heat shock, chemicals, light, etc.) which is different from the level of transcription of the operably linked nucleic acid sequence in the absence of the stimulus.
  • a promoter sequence functioning in bateria is understood as meaning, in principle, any promoter which is capable of governing the expression of genes, in particular foreign genes, in bacteria cells.
  • expression can be, for example, constitutive, inducible or development-dependent.
  • a constitutive promoter is a promoter where the rate of RNA polymerase binding and initiation is approximately constant and relatively independent of external stimuli.
  • Usable promoters are constitutive promoters, such as cos, tac, trp, tet, trp-tet, Ipp, lac, Ipp-lac, lacli' T7, T5, T3, gal, trc, ara, SP6, ⁇ -PR or in the ⁇ -P ⁇ _ promoter, all of which are advantageously used in Gram-negative bacteria.
  • Other advantageous regulatory sequences are contained, for example, in the Gram-positive promoters amy and SPO2, in the yeast or fungal promoters ADC1 , MFa , AC, P-60, CYC1 , GAPDH, TEF, rp28, ADH.
  • all natural bacterial promoters with their regulatory sequences as those mentioned above may be used for the process according to the invention.
  • synthetic promoters may also advantageously be used.
  • Polypeptide, peptide, oligopeptide, gene product, expression product and protein are used interchangeably herein to refer to a polymer or oligomer of consecutive amino acid residues.
  • Recombinant or transgenic DNA expression construct with respect to, for example, a nucleic acid sequence (expression construct, expression cassette or vector comprising said nucleic acid sequence) refers to all those constructs originating by experimental manipulations in which either a) said nucleic acid sequence, or b) a genetic control sequence linked operably to said nucleic acid sequence (a), for example a promoter, or c) (a) and (b) is not located in its natural genetic environment or has been modified by experimental manipulations, an example of a modification being a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues.
  • Natural genetic environment refers to the natural chromosomal locus in the organism of origin, or to the presence in a genomic library.
  • the natural genetic environment of the nucleic acid sequence is preferably retained, at least in part.
  • the environment flanks the nucleic acid sequence at least at one side and has a sequence of at least 50 bp, preferably at least 500 bp, especially preferably at least 1 ,000 bp, very especially preferably at least 5,000 bp, in length.
  • a naturally occurring expression construct - for example the naturally occurring combination of a promoter with the corresponding gene - becomes a transgenic expression construct when it is modified by non-natural, synthetic "artificial" methods such as, foexample, mutagenesis.
  • Recombinant polypeptides or proteins refer to polypeptides or proteins produced by recombinant DNA techniques, i.e., produced from cells transformed by an exogenous recombinant DNA construct encoding the desired polypeptide or protein.
  • Recombinant nucleic acids and polypeptide may also comprise molecules which as such does not exist in nature but are modified, changed, mutated or otherwise manipulated by man.
  • the recombinant DNA expression construct confers expression of one or more nucleic acid molecules.
  • Said recombinant DNA expression construct according to the invention advantageously encompasses a promoter functioning in bacteria, additional regulatory or control elements or sequences functioning in bacteria and a terminator functioning in bacteria. Additionally, the recombinant expression construct might contain additional functional elements such as expression cassettes conferring expression of e.g. positive and negative selection markers, reporter genes, recombinases or endonucleases effecting the production, amplification or function of the expression cassettes, vectors or recombinant organisms according to the invention.
  • additional functional elements such as expression cassettes conferring expression of e.g. positive and negative selection markers, reporter genes, recombinases or endonucleases effecting the production, amplification or function of the expression cassettes, vectors or recombinant organisms according to the invention.
  • the recombinant expression construct can comprise nucleic acid sequences homologous to a bacterial gene of interest having a sufficient length in order to induce a homologous recombination (HR) event at the locus of the gene of interest after introduction in the bacteria.
  • HR homologous recombination
  • a recombinant transgenic expression cassette of the invention (or a transgenic vector comprising said transgenic expression cassette) can be produced by means of customary recombination and cloning techniques as are described (for example, in Maniatis 1989, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor (NY); Silhavy 1984, ) Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; and in Ausubel 1987, Current Protocols in Molecular Biology, Greene Publishing Assoc, and Wiley Interscience).
  • the introduction of an expression cassette according to the invention into a bacteria can be effected advantageously using vectors, which comprise the above described nucleic acids, promoters, terminators, regulatory or control elements and functional elements.
  • Regulatory sequence refers to promoters, enhancer or other segments of DNA where regulatory proteins such as transcription factors bind and thereby influencing the transcription rate of a given gene.
  • rumen bacteria refers to those bacteria that can be isolated from the rumen or gastrointestinal tract of ruminant animals (sheep, goats, cattle, deer, etc) where a large part of their digestive process is performed by bacteria.
  • Structural gene as used herein is intended to mean a DNA sequence that is transcribed into mRNA which is then translated into a sequence of amino acids characteristic of a specific polypeptide.
  • Transforming or transformation refers to the introduction of genetic material (e.g., a transgene) into a cell. Transformation of a cell may be stable or transient.
  • transient transformation or “transiently transformed” refers to the introduction of one or more transgenes into a cell in the absence of integration of the transgene into the host cell's genome.
  • transient transformant refers to a cell which has transiently incorporated one or more transgenes.
  • stable transformation or “stably transformed” refers to the introduction and integration of one or more transgenes into the genome of a cell, preferably resulting in chromosomal integration and stable heritability.
  • Stable transformation of a cell may be detected by Southern blot hybridization of genomic DNA of the cell with nucleic acid sequences, which are capable of binding to one or more of the transgenes.
  • stable transformation of a cell may also be detected by the polymerase chain reaction of genomic DNA of the cell to amplify transgene sequences.
  • the term "stable transformant" refers to a cell that has stably integrated one or more transgenes into the genomic DNA.
  • a stable transformant is distinguished from a transient transformant in that, whereas genomic DNA from the stable transformant contains one or more transgenes, genomic DNA from the transient transformant does not contain a transgene. Transformation also includes introduction of genetic material into bacteria cells in the form of vectors involving extrachromosomal replication and gene expression. These vectors can be replicated autonomously in the host organism.
  • Transgenic or recombinant when used in reference to a cell refers to a cell which contains a transgene, or whose genome has been altered by the introduction of a transgene.
  • Transgenic cells may be produced by several methods including the introduction (as defined above) of a "transgene” comprising nucleic acid (usually DNA) into a target cell or integration of the transgene into a chromosome of a target cell by way of human intervention, such as by the methods described herein.
  • a transgene comprising nucleic acid (usually DNA) into a target cell or integration of the transgene into a chromosome of a target cell by way of human intervention, such as by the methods described herein.
  • the skilled worker can find sutiable methods in the following publications:
  • paracasei NFBC 338 cells were prepared using 3.5X SMEB (1 M sucrose, 3.5mM MgCI ⁇ ) as described by Luchansky et al. (1988). Sequence analysis was performed using DNAStar software (DNAStar, Madison, Wisconsin, USA), de Ruyter, P. G., O. P. Kuipers, and W. M. de Vos. 1996. Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin. Appl Environ
  • Treatment refers to the therapeutical application of a medicament comprising fermentative oil, preferably purified conjugated linoleic acid, more preferably trans-10, cis 12 octadecadienoic acid produced using the inventive process.
  • Said therapeutical application is to be understood in a broad sense and comprises e.g. the application of said medicament in order to (i) prevent the formation of cancer cells, (ii) reduce or stop the growth of cancer cells and/or (iii) prevent the spread of cancer cells throughout the body
  • Wild-type, natural or of natural origin means with respect to an organism, polypeptide, or nucleic acid sequence, that said organism polypeptide, or nucleic acid sequence is naturally occurring or available in at least one naturally occurring organism polypeptide, or nucleic acid sequence which is not changed, mutated, or otherwise manipulated by man.
  • Vector is a DNA molecule capable of replication in a host cell. Plasmids and cosmids are exemplary vectors. Furthermore, the terms “vector” and “vehicle” are used interchangeably in reference to nucleic acid molecules that transfer DNA segment(s) from one cell to another, whereby the cells not necessarily belonging to the same organism (e.g. transfer of a DNA segment form an Agrobacterium cell to a plant cell).
  • expression vector refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism.
  • the teaching of the present invention enables the production of trans-10, cis 12 octadecadienoic acid in transgenic microorganism.
  • a first embodiment of the present invention relates to a process for the production of trans-10, cis 12 conjugated linoleic acid in a transgenic microorganism comprising the steps of:
  • the invention relates to a process for the production of conjugated linoleic acid in a transgenic microorganism according to the above described steps (a) to (e), characterized in that the produced conjugated linoleic acid is a mixture of different CLA isoforms comprising at least 30%, preferably at least 40%, more preferably at least 50%, especially preferably at least 60%, very especially preferably at least 70%, most preferably at least 80% of trans-10, cis 12 octadecadienoic acid.
  • the isomeric purity of the trans-10, cis 12 octadecadienoic acid can advantageously be further increased by methods known to the skilled artisian e.g. crystallization.
  • nucleic acid molecule introduced into the microorganism as described under (a) encodes for a polypeptide with conjugated linoleic acid isomerase activity which is able to convert linoleic acid (9 cis, 12 cis- octadecadienoic acid) to trans-10, cis 12 octadecadienoic acid and can be selected from the following:
  • nucleic acid molecule having the sequence as described in SEQ ID No. 1 , or ii. from functional equivalents of the polypeptide encoded by the nucleic acid molecule described in (i) such as: a. a nucleic acid molecule having at least 50, preferably at least 75, more preferably at least 100, especially preferably at least 125, very especially preferably at least 150 consecutive base pairs of the sequence described by SEQ ID No.1 , or b.
  • nucleic acid molecule having an identity of at least 80%, preferably at least 85%, more preferably at least 90%, especially preferably at least 95%, very especially preferably at least 98% over a sequence of at least 100, preferably at least 125, more preferably at least 150, especially preferably at least 175, very especially preferably at least 200 consecutive nucleic acid base pairs to the sequence described by SEQ ID No. 1 , or c.
  • a nucleic acid molecule hybridizing under high stringent conditions with a nucleic acid fragment of at least 50, preferably at least 100, more preferably at least 150, especially preferably at least 200, very especially preferably at least 500 consecutive base pairs of a nucleic acid molecule described by SEQ ID No.
  • nucleic acid molecule encoding a polypeptide having at least 75%, preferably at least 85%, more preferably at least 90%, especially preferably at least 95%, very especially preferably at least 98% identity to the amino acid sequence as shown in SEQ ID No. 2.
  • nucleic acid sequences as defined in ((i) and (ii)) can in principle be identified and isolated from all microorganisms.
  • SEQ ID No. 1 or its homologs/functional equivalents can advantageously be isolated from bacteria, preferrably those bacteria able to produce conjugated fatty acids.
  • Bacteria which may be mentioned are Gram-negative and Gram-positive bacteria.
  • nucleic acid molecules according to the invention are preferably isolated by methods known to the skilled worker from Gram-positive bacteria such as Propionibacterium, Lactococcus, Bifidobacterium or Lactobacillus, advantageously from Bifidobacterium.
  • Functional derivatives of the sequence given in SEQ ID No.1 are furthermore to be understood as meaning, for example, allelic variants having at least 75%, preferably at least 85%, more preferably at least 90%, especially preferably at least 95%, very especially preferably at least 98% identity.
  • the identity was calculated as described in the general defintions or by using additional computer programs like PiIeUp (J. MoI. Evolution., 25 (1987), 351-360, Higgins et al., CABIOS, 5 1989: 151-153).
  • the amino acid sequence derived from the above-mentioned nucleic acid is described by sequence SEQ ID No. 2.
  • Allelic variants encompass, in particular, functional variants which can be obtained from the sequence shown in SEQ ID No. 1 by means of deletion, insertion or substitution of nucleotides, the enzymatic activity of the derived synthetic proteins being retained.
  • Functional equivalents of the above-described conjugated linoleic acid isomerase can be identified via homology searches in nucleic acid databases or via DNA hybridization (screening of genomic DNA libraries) using a fragment of at least 50 preferably at least 100, more preferably at least 150, especially preferably at least 200, very especially preferably at least 500 consecutive base pairs of the nucleic acid molecule described by the SEQ ID No. 1 and stringent hybridization conditions.
  • the stringent hybridizing conditions can be chosen as follows:
  • the hybridization puffer contains Formamide, NaCI and PEG 6000 (Polyethyleneglykol MW 6000).
  • Formamide has a destabilizing effect on double strand nucleic acid molecules, thereby, when used in hybridization buffer, allowing the reduction of the hybridization temperature to 42°C without reducing the hybridization stringency.
  • NaCI has a positive impact on the renaturation-rate of a DNA duplex and the hybridization efficiency of a DNA probe with its complementary DNA target.
  • PEG increases the viscosity of the hybridization buffer, which has in principle a negative impact on the hybridization efficiency.
  • the composition of the hybridization buffer is as follows:
  • hybridization is preferably performed over night at 42°C. In the morning, the hybridized filter will be washed 3 x for 10 minutes with 2xSSC + 0,1 % SDS.
  • Hybridization should advantageously be carried out with fragments of at least 50, 60, 70 or 80 bp, preferably at least 90 bp. In an especially preferred embodiment, the hybridization should be carried out with the entire nucleic acid sequence with conditions described above.
  • amino acid sequences according to the invention are to be understood as meaning proteins which contain an amino acid sequence shown in SEQ ID No. 2 or a sequence obtainable therefrom by the substitution, inversion, insertion or deletion of one or more amino acid residues, the enzymatic activity of the protein shown in SEQ ID No. 2 being retained or not reduced substantially.
  • the term not reduced substantially is to be understood as meaning all enzymes which still have at least 10%, preferably 20%, especially preferably 30% of the enzymatic activity of the starting enzyme.
  • certain amino acids may be replaced by others with similar physico-chemical properties (spatial dimension, basicity, hydrophobicity and the like).
  • arginine residues are exchanged for lysine residues, valine residues for isoleucine residues or aspartic acid residues for glutamic acid residues.
  • valine residues for isoleucine residues
  • aspartic acid residues for glutamic acid residues.
  • trans-10, cis-12 conjugated linoleic acid isomerase is isolated from a rumen bacteria, preferably from Megashera elsdenii YJ-4.
  • nucleic acid molecule encoding trans-10, cis-12 conjugated linoleic acid isomerase is isolated from a Propionibacterium, preferably from Propionibacterium acnes.
  • trans-10, cis-12 conjugated linoleic acid isomerase is the trans-10, cis-12 conjugated linoleic acid isomerase with the accession no. CQ766028 isolated from Propionibacterium acnes (SEQ ID No. 1).
  • the above described nucleic acid molelcule is part of an recombinant or transgenic DNA expression construct (as defined in the general definitons).
  • the recombinant or transgenic DNA expression construct is to be understood as meaning the sequence given in SEQ ID No. 1 , or functional equivalents of the polypeptide encoded by the nucleic acid molecule described by SEQ ID No. 1 (as defined above in (H)) which have been linked functionally to one or more regulatory signals, advantageously for increasing gene expression).
  • These regulatory sequences are, for example, sequences to which inductors or repressors bind and thus regulate the expression of the nucleic acid.
  • the natural regulation of these sequences upstream of the actual structural genes may still be present and, if desired, may have been genetically altered in such a way that the natural regulation has been switched off and the expression of the genes increased.
  • the expression of the gene construct may also have a simpler structure, viz. no additional regulatory signals have been inserted upstream of the sequence or its derivatives and the natural promoter with its regulation has not been removed. Instead, the natural regulatory sequence has been mutated in such a way that regulation no longer takes place and gene expression is increased.
  • These altered promoters may also be placed upstream of the natural gene on their own, in order to increase activity.
  • the gene construct can also advantageously contain one or more so-called enhancer sequences functionally linked to the promoter, and these allow an increased expression of the nucleic acid sequence. It is also possible to insert, at the 3' end of the DNA sequences, additional advantageous sequences such as further regulatory elements or terminators. One or more copies of the conjugated linoleic acid isomerase gene may be contained in the gene construct.
  • Advantageous regulatory sequences for the process according to the invention are contained, for example, in promoters such as cos, tac, trp, tet, trp-tet, Ipp, lac, Ipp-lac, lacli' T7, T5, T3, gal, trc, ara, SP6, ⁇ -PR or in the ⁇ -P ⁇ _ promoter, all of which are advantageously used in Gram- negative bacteria.
  • promoters such as cos, tac, trp, tet, trp-tet, Ipp, lac, Ipp-lac, lacli' T7, T5, T3, gal, trc, ara, SP6, ⁇ -PR or in the ⁇ -P ⁇ _ promoter, all of which are advantageously used in Gram- negative bacteria.
  • Other advantageous regulatory sequences are contained, for example, in the Gram-positive promoters amy and SPO2, in the yeast or fungal promoters ADC1 , MFa, AC, P- 60, CY
  • said recombinant or transgenic DNA expression construct advantageously contains, for expression of the genes present, in addition 3' and/or 5' terminal regulatory sequences to increase expression, these being selected for optimal expression depending on the selected host organism and gene or genes.
  • regulatory sequences are intended to make specific gene expression possible. This may mean, for example depending on the host organism, that the gene is expressed or overexpressed only after induction, or that it is expressed and/or overexpressed immediately.
  • the regulatory sequences or factors may for this purpose preferably have a beneficial effect on expression of the introduced genes, and thus increase it.
  • an enhancement of the regulatory elements can advantageously take place at the level of transcription, by using strong transcription signals such as promoters and/or enhancers.
  • strong transcription signals such as promoters and/or enhancers.
  • the recombinant or transgenic DNA expression construct may also contain further genes to be introduced into organisms. These genes can be under separate regulation or under the same regulatory region as the isomerase gene according to the invention. These genes are, for example, other biosynthesis genes, advantageously of the fatty acid and lipid biosynthesis, which allow increased synthesis of the isomerase starting material such as linoleic acid.
  • the nucleic acid sequences For optimal expression of heterologous genes in organisms it is advantageous to modify the nucleic acid sequences in accordance with the specific codon usage of the organism.
  • the codon usage can easily be established on the basis of computer analyses of other, known genes of the relevant organism.
  • the nucleic acid fragment For expression in a host organism, for example a microorganism such as yeasts or bacteria, the nucleic acid fragment is advantageously inserted into a vector such as, for example, a plasmid, a phage or other DNA, which vector allows optimal expression of the genes in the host.
  • suitable plasmids are, in E.
  • coli pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1 , pHS2, pPLc236, pMBL24, pLG200, pUR290, plN-ll
  • the plasmids mentioned represent a small selection of the plasmids which are possible.
  • Other plasmids are well known to the skilled worker and can be found, for example, in the book Cloning Vectors (Eds. Pouwels P. H. et al. Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018).
  • Suitable plant vectors are described, inter alia, in "Methods in Plant Molecular Biology and Biotechnology" (CRC Press), Chapter 6/7, pp.71-119.
  • vectors are also to be understood as meaning all the other vectors which are known to the skilled worker, such as, for example, phages, IS elements, linear or circular DNA. These vectors can be replicated autonomously in the host organism or replicated chromosomally. Autonomous replication is preferred.
  • the vector advantageously contains at least one copy of the nucleic acid sequence according to the invention.
  • the nucleic acid fragment advantageously additionally contains 3'- and/or 5'-terminal regulatory sequences to increase expression, these sequences being selected for optimal expression, depending on the host organism chosen and the gene or genes.
  • regulatory sequences should allow the targeted expression of the gene. Depending on the host organism, this may mean, for example, that the gene is expressed and/or overexpressed only after induction, or that it is expressed and/or overexpressed immediately.
  • the regulatory sequences or factors can preferably have a positive effect on, and thus increase, the gene expression of the genes introduced.
  • strengthening of the regulatory elements can advantageously take place at the transcriptional level by using strong transcription signals such as promoters and/or enhancers.
  • strengthening of translation is also possible, for example by improving mRNA stability.
  • the gene construct according to the invention can advantageously also be introduced into the organisms in the form of a linear DNA and integrated into the genome of the host organism by means of heterologous or homologous recombination.
  • This linear DNA may consist of a linearized plasmid or only of the nucleic acid fragment as vector or of the nucleic acid sequence according to the invention.
  • the nucleic acid sequence according to the invention is advantageously cloned into a nucleic acid construct together with at least one reporter gene, and the nucleic acid construct is introduced into the genome.
  • This reporter gene should allow easy detectability via a growth assay, a fluorescence assay, a chemo assay, a bioluminescence assay or a resistance assay, or via a photometric measurement. Examples of reporter genes which may be mentioned are genes for resistance to antibiotics (e.g.
  • amplicilin, chloramphenicol, Tetracyclin, erythromycin) or hydrolase genes fluorescence protein genes, bio-luminescence genes, sugar metabolism genes or nucleotide metabolism genes, or biosynthesis genes such as the Ura3 gene, the Ilv2 gene, the luciferase gene, the ⁇ -galactosidase gene, the gfp gene, the 2-deoxyglucose-6- phosphate phosphatase gene, the ⁇ -glucuronidase gene, the ⁇ -lactamase gene, the neomycin phospho-transferase gene or the hygromycin phosphotransferase gene
  • nucleic acid sequence according to the invention may also be introduced into an organism on its own.
  • nucleic acid according to the invention can be introduced into organisms, for example bacteria, by methods known to the skilled worker.
  • Suitable organisms or host organisms (transgenic organisms) for the process according to the invention are, in principle, all organisms which are capable of synthesizing unsaturated fatty acids, and which are suitable for the expression of recombinant genes.
  • Examples which may be mentioned belong to the family selected from the group consiting of Lactobacillaceae, Streptococcaceae, Propionibacteriaceae, Enterobacteriaceae and Bifidobacteriaceae, preferably to the genus selected from the group consiting of Lactococcus, Lactobacillus, Propionibacterium, Escherichia and Bifidobacterium, most preferably said microorganism is selected from group consisting of Lactococcus lactis, Lactobacillus paracasei and Escherichia coli.
  • nucleic acid molecule sources include in general all prokaryotic or eukaryotic cells, preferably unicellular microorganisms, such as fungi like the genus Claviceps or Aspergillus or gram-positive bacteria such as the genera Bacillus, Corynebacterium, Micrococcus, Brevibacterium, Rhodococcus, Nocardia, Caseobacter or Arthrobacter or gram- negative bacteria such as the genera Escherichia, Flavobacterium or Salmonella, or yeasts such as the genera Rhodotorula, Hansenula or Candida.
  • fungi like the genus Claviceps or Aspergillus or gram-positive bacteria such as the genera Bacillus, Corynebacterium, Micrococcus, Brevibacterium, Rhodococcus, Nocardia, Caseobacter or Arthrobacter or gram- negative bacteria such as the genera Escherichia, Flavobacterium or Salmonella, or yeasts such as the genera Rhodot
  • Production strains which are especially advantageously selected in the process according to the invention are microorganisms selected from the group of the families Actinomycetaceae, Bacillaceae, Brevibacteriaceae, Corynebacteriaceae, Enterobacteriacae, Gordoniaceae, Micrococcaceae, Mycobacteriaceae, Nocardiaceae, Pseudomonaceae, Rhizobiaceae, Streptomycetaceae, Chaetomiaceae, Choanephoraceae, Cryptococcaceae, Cunninghamellaceae, Demetiaceae, Moniliaceae, Mortierellaceae, Mucoraceae, Pythiaceae, Sacharomycetaceae, Saprolegniaceae, Schizosacharomycetaceae, Sodariaceae,
  • microorganisms are grown in a liquid medium which contains a carbon source, usually in the form of sugars, a nitrogen source, usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, a phosphate source such as potassium hydrogen phosphate, trace elements such as iron salts, manganese salts, magnesium salts and, if required, vitamins, at temperatures between 0 0 C and 100 0 C, preferably between 10 0 C and 60 0 C, more preferably between 15°C and 50°C, while gassing in oxygen.
  • a carbon source usually in the form of sugars
  • a nitrogen source usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate
  • a phosphate source such as potassium hydrogen phosphate
  • trace elements such as iron salts, manganese salts, magnesium salts and, if required, vitamins, at temperatures between 0 0 C and 100 0 C, preferably between 10 0 C and 60 0
  • the pH of the liquid medium can be maintained at a fixed value, i.e. the pH is regulated while culture takes place.
  • the pH should then be in a range between pH 2 and pH 9.
  • the microorganisms may also be cultured without pH regulation. Culturing can be effected by the batch method, the semi-batch method or fed-batch/continuously. Nutrients may be supplied at the beginning of the fermentation or fed in semicontinuously or continuously.
  • the organism can be grown under aerobic or anaerobic conditions.
  • the pH of the liquid medium can be maintained at a fixed value, i.e. the pH is regulated while culture takes place.
  • the pH should then be in a range between pH 2 and pH 9, preferably between 4 and 8.5, 4.5 and 8, more preferably between 5 and 7.5, 5.5 and 7.
  • the microorganisms may also be cultured without pH regulation
  • the process according to the invention is advantageously carried out at temperatures between 0 0 C and 100 0 C, preferably between 10 0 C and 65°C, 15°C and 55°C, more preferably between 20 0 C and 50 0 C, 25°C and 45°C, particularly preferred between 30 0 C and 40°C while gassing in oxygen.
  • the pH in the process (in vitro) according to the invention is advantageously kept between pH 4 and 12, preferably between 4 and 8.5, 4.5 and 8, more preferably between 5 and 7.5, 5.5 and 7.
  • the microorganisms may also be cultured without pH regulation.
  • a summary of known cultivation methods is to be found in the textbook by Chmiel (Bioproze ⁇ technik 1. Einf ⁇ hrung in die Biovonstechnik (Gustav Fischer Verlag, Stuttgart, 1991 )) or in the textbook by Storhas (Bioreaktoren und periphere bamboo (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).
  • the culture medium to be used must meet the requirements of the respective strains in a suitable manner.
  • culture media for various microorganisms are present in the handbook "Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D. C, USA, 1981).
  • These media which can be employed according to the invention include, as described above, usually one or more carbon sources, nitrogen sources, inorganic salts, vitamins and/or trace elements.
  • Preferred carbon sources are sugars such as mono-, di- or polysaccharides. Examples of very good carbon sources are glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose.
  • Sugars can also be added to the media via complex compounds such as molasses, or other byproducts of sugar refining. It may also be advantageous to add mixtures of various carbon sources.
  • Other possible carbon sources are oils and fats such as, for example, soybean oil, sunflower oil, peanut oil and/or coconut fat, fatty acids such as, for example, palmitic acid, stearic acid and/or linoleic acid, alcohols and/or polyalcohols such as, for example, glycerol, methanol and/or ethanol and/or organic acids such as, for example, acetic acid and/or lactic acid.
  • Nitrogen sources are usually organic or inorganic nitrogen compounds or materials, which contain these compounds.
  • nitrogen sources include ammonia in liquid or gaseous form or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources such as corn steep liquor, soybean meal, soybean protein, yeast extract, meat extract and others.
  • the nitrogen sources may be used singly or as a mixture.
  • Inorganic salt compounds, which may be present in the media include the chloride, phosphorus or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.
  • phosphorus source phosphoric acid potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts.
  • Chelating agents can be added to the medium in order to keep the metal ions in solution.
  • Particularly suitable chelating agents include dihydroxyphenols such as catechol or protocatechuate, or organic acids such as citric acid.
  • the fermentation media employed according to the invention for cultivating microorganisms normally also contain other growth factors such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenate and pyridoxine.
  • Growth factors and salts are often derived from complex media components such as yeast extract, molasses, corn steep liquor and the like. Suitable precursors can moreover be added to the culture medium. The exact composition of the media compounds depends greatly on the particular experiment and is chosen individually for each specific case. Information about media optimization is obtainable from the textbook "Applied Microbiol. Physiology, A Practical Approach” (editors P.M. Rhodes, P. F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). Growth media can also be purchased from commercial suppliers such as Standard 1 (Merck) or BHI (Brain heart infusion, DIFCO) and the like.
  • Standard 1 Merck
  • BHI Brain infusion
  • All media components are sterilized either by heat (1.5 bar and 121 0 C for 20 min) or by sterilizing filtration.
  • the components can be sterilized either together or, if necessary, separately.
  • All media components can be present at the start of the cultivation or optionally be added continuously or batchwise.
  • the temperature of the culture is normally between 15°C and 45°C, preferably at 25°C to 40 0 C, and can be kept constant or changed during the experiment.
  • the pH of the medium should be in the range from 5 to 8.5, preferably around 7.
  • the pH for the cultivation can be controlled during the cultivation by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia or acidic compounds such as phosphoric acid or sulfuric acid.
  • Foaming can be controlled by employing antifoams such as, for example, fatty acid polyglycol esters.
  • the stability of plasmids can be maintained by adding to the medium suitable substances having a selective effect, for example antibiotics. Aerobic conditions are maintained by introducing oxygen or oxygen-containing gas mixtures such as, for example, ambient air into the culture.
  • the temperature of the culture is normally from 20 0 C to 45°C and preferably from 25°C to 40 0 C.
  • the culture is continued until formation of the desired product is at a maximum. This aim is normally achieved within 10 hours to 160 hours.
  • Disrupted cells mean, for example, cells which have been made permeable by treatment with, for example, solvents, or cells which have been ruptured by an enzyme treatment, by a mechanical treatment (for example French press or ultrasound) or by another method.
  • the crude extracts obtained in this way are advantageously suitable for the process according to the invention.
  • Purified or partially purified enzymes can also be used for the process.
  • immobilized microorganisms or enzymes which can advantageously be used in the reaction.
  • Linoleic acid as a major starting material can be added to the reaction mixture batchwise, semibatchwise or continuously.
  • concentration of the starting material for the fermentation process which is preferably linoleic acid is not higher than 3 mg/ml, preferably not higher than 2 mg/ml, more preferably not higher than 1 mg/ml, especially preferably not higher than 0.5 mg/ml.
  • the concentration of linoleic acid used to induce the production of trans-10, cis 12 octadecadienoic acid is ranging from 0.1 to 0.5 mg/ml, preferrably from 0.4 to 0.5 mg/ml, more preferrably from 0.3 to 0.4 mg/ml, especially preferrably from 0.2 to 0.3 mg/ml, most preferably from 0.1 to 0.2 mg/ml.
  • the linoleic acid is added to a micororganism culture having an optical density (OD ⁇ oo) of at least 0.1 , preferably of at least 0.2, more preferably of at least 0.3, or 0.31 , 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, especially preferably of at least 0.4, or 0.41 , 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, very especially preferably of at least 0.5, or 0.51 , 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6.
  • the linoleic acid can even be added to micororganism cultures having an optical density (OD ⁇ oo) above 0,6.
  • the induced culture prior to isolation of the CLA, is incubated for at least 12 to 18 hours, preferrably for at least 18 to 24 hours, more preferably for at least 24 to 30 hours, especially preferably for at least 30 to 42 hours, most preferably for at least 42 to 72 hours.
  • the products have a high isomeric purity, which can advantageously be further increased where necessary by the crystallization.
  • the inventive process leads to trans-10, cis 12 octadecadienoic acid as major product.
  • the fatty acids produced can be isolated from the organism by methods with which the skilled worker is familiar. For example via extraction, salt precipitation and/or different chromatography methods. In the case of the fermentation of microorganisms, the abovementioned fatty acids may accumulate in the medium and/or the cells. If microorganisms are used in the process according to the invention, the fermentation broth can be processed after the cultivation. Depending on the requirement, all or some of the biomass can be removed from the fermentation broth by separation methods such as, for example, centrifugation, filtration, decanting or a combination of these methods, or else the biomass can be left in the fermentation broth.
  • the fermentation broth can subsequently be reduced, or concentrated, with the aid of known methods such as, for example, rotary evaporator, thin-layer evaporator, falling film evaporator, by reverse osmosis or by nanofiltration. Afterwards advantageously further compounds for formulation can be added such as corn starch or silicates.
  • This concentrated fermentation broth advantageously together with compounds for the formulation can subsequently be processed by lyophilization, spray drying, spray granulation or by other methods.
  • the fatty acids or the fatty acid compositions are isolated from the organisms, such as the microorganisms or the culture medium in or on which the organisms have been grown, or from the organism and the culture medium, in the known manner, for example via extraction, distillation, crystallization, chromatography or a combination of these methods.
  • These purification methods can be used alone or in combination with the aforementioned methods such as the separation and/or concentration methods.
  • the product-containing composition can be subjected for example to a thin layer chromatography on silica gel plates or to a chromatography such as a Florisil column (Bouhours J. F., J. Chromatrogr. 1979, 169, 462), in which case the desired product or the impurities are retained wholly or partly on the chromatography resin.
  • chromatography steps can be repeated if necessary, using the same or different chromatography resins.
  • the skilled worker is familiar with the choice of suitable chromatography resins and their most effective use.
  • An alternative method to purify the fatty acids is for example crystallization in the presence of urea. These methods can be combined with each other. The identity and purity of the isolated compound(s) can be determined by prior art techniques.
  • the invention relates to a process for the production of conjugated linoleic acid in a transgenic microorganism according to the above described steps (a) to (e), characterized in that the bioconversion rate (as defined in the general definitions) of linoleic acid in the operation or fermentation procedure (batch or fed-batch) is higher than 10%, preferably higher than 20%, more preferably higher than 30% for bacteria belonging to the genus Lactobacillus, higher than 10%, preferably higher than 20%, more preferably higher than 30%, especially preferably higher than 40% for bacteria belonging to the genus Escherichia and higher than 10%, preferably higher than 20%, more preferably higher than 30%, especially preferably higher than 40%, very especially preferably higher than 50% for bacteria belonging to the genus Lactococcus.
  • the invention relates to a process for the production of feed or food products or nutraceuticals enriched in conjugated linoleic acid, wherein the conjugated linoleic acid is produced according to the above described process.
  • the invention relates furthermore to feed-, food-products and nutraceuticals enriched in conjugated linoleic acid, wherein the conjugated linoleic acid is produced according to the above described process.
  • compositions of the present invention find a wide variety of nutritional, therapeutic and pharmacological uses. These uses include: the reduction of body fat in animals: increasing muscle mass in animals, increasing feed efficiency in animals, reducing body weight in humans, attenuating allergic reactions in animals, preventing weight loss due to immune stimulation in animals, increasing the mineral content of bone in animals, preventing skeletal abnormalities in animals, and decreasing the amount of cholesterol in the blood of animals.
  • the feed- or food-products preferably preparations used as additives for feed- or food- products, in addition to the conjugated linoleic acid, preferably the fementated oil or the purified conjugated linoleic acid isomer mixture, more preferrably the purified trans-10, cis 12 octadecadienoic acid, produced according to the above described process, can comprise further constituents.
  • the choice of further constituents will be guided here by the chosen field of use of the preparations and is in general known to the skilled artisian.
  • constituents within the meaning of the present invention which come into consideration are, for example, the following substances: further organic acids, carotenoids, trace elements, antioxidants, vitamins, enzymes, amino acids, minerals, emulsifiers, stabilizers, preservatives, anticaking agents and/or flavor enhancers.
  • Organic acids which are preferably used are formic acid, propionic acid, lactic acid, acetic acid and citric acid, particular preference being given to formic acid, propionic acid or lactic acid.
  • carotenoids are taken to mean tetraterpenes in which one or two ionone rings are bonded by a carbon chain having 9 double bonds and can be of either plant or animal origin. Carotenoids are also taken to mean the oxygenated xanthophylls.
  • inventive preparations can comprise, for example, the following trace elements: chromium, iron, fluorine, iodine, cobalt, copper, manganese, molybdenum, nickel, selenium, vanadium, zinc or tin.
  • Antioxidants which can be used are, for example, ascorbic acid (vitamin C, E 300), sodium L- ascorbate (E 301), calcium L-ascorbate (E 302), ascorbyl palmitate (E 304), butylated hydroxyanisole (E 320), butylated hydroxytoluene (E 321), calcium disodium EDTA (E 385), gallates, for example propyl gallate (E 310), octyl gallate (E 311), dodecyl gallate (lauryl gallate) (E 312), isoascorbic acid (E 315), sodium isoascorbate (E 316), lecithin (E 322), lactic acid (E 270), multiple phosphates, for example diphosphates (E 450), triphosphates (E 451 ), polyphosphates (E 452), sulfur dioxide (E 220), sodium sulfite (E 221 ), sodium bisulfite (E 222
  • Vitamins which come into consideration are not only fat-soluble vitamins, but also water-soluble vitamins.
  • fat-soluble vitamins are: vitamin A (retinol), vitamin D (calciferols), vitamin E (tocopherols and tocotrienols), vitamin K (phylloquinones and menaquinones), preference being given to vitamins A and E.
  • water-soluble vitamins are: vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxin), vitamin B12 (cobalamin), vitamin C (ascorbic acid), vitamin H (biotin), folic acid and niacin, preference being given to the vitamins B2 and C.
  • the preparations can also comprise enzymes. Those which may be mentioned by way of example are: amylases, proteases and invertases.
  • Amino acids coming into consideration in the context of this invention are, for example, glutamic acid, L-carnitine, L-glutamine, L-taurine, L-aspartic acid, L-glycine, L-lysine, DL-phenylalanine, L-tryptophan, tyrosine, L-arginine, L-cysteine, L-leucine, L-methionine, L-alanine, L-serine, L- threonine, L-citrulline, L-valine, L-histidine, L-isoleucine, L-ornithine or L-proline.
  • amino acids for example L-isoleucine, L-leucine, L-lysine, L-methionine, DL-phenylalanine, L-threonine, L-tryptophan and L-valine, very particular preference being given to the amino acids important in animal nutrition L-lysine, DL-methionine or L-threonine.
  • Minerals in the context of this invention are, for example, sodium, potassium, magnesium, calcium, phosphorus, iron and zinc.
  • emulsifiers use can be made of the following substances, for example:
  • E 482 calcium stearoyl-2-lactylate, E 483 stearyl tartrate, E 491 sorbitan monostearate, E 492 sorbitan tristearate, E 493 sorbitan monolaurate, E 494 sorbitan monooleate or E 495 sorbitan monopalmitate.
  • Stabilizers are substances which maintain the consistency or the composition of foods. Those which may be mentioned by way of example are: ascorbic acid (E 300), carbamide (E 927b), iron(ll) lactate (E 585), iron gluconate (E 579), glycerol esters (E 445), lecithin (E 322), metatartaric acid (E 353), pectin (E 440), sucrose acetate isobutyrate (E 444) and tin(ll) chloride (E 512).
  • Preservatives are substances which prolong the shelf life of foods, by protecting them from the harmful effects of microorganisms. Those which may be mentioned by way of example are: E 200 sorbic acid, E 201 sodium sorbate, E 202 potassium sorbate, E 203 calcium sorbate, E 210 benzoic acid, E 211 sodium benzoate, E 212 potassium benzoate, E 213 calcium benzoate, E 214 ethyl p-hydroxybenzoate/PHB ester, E 215 sodium ethyl p- hydroxybenzoate/PHB ethyl ester sodium salt, E 216 propyl p-hydroxybenzoate/PHB propyl ester, E 217 sodium propyl p-hydroxybenzoate/PHB-propyl ester sodium salt, E 218 methyl p- hydroxybenzoate/PHB-methyl ester, E 219 sodium methyl p-hydroxybenzoate/PHB-methyl ester sodium salt, E 220 sulfur dioxide, E 2
  • Anticaking agents in the context of the present invention are naturally occurring or synthesized substances which increase the flowability of a food by preventing the clumping together and sticking together of the particles.
  • Examples which may be mentioned are: E 530 magnesium oxide, E 535 sodium ferrocyanide, E 536 potassium ferrocyanide, E 541 acidic sodium aluminum phosphate, E 551 silicon dioxide, E 552 calcium silicate, E 553ai magnesium silicate, E 553aii magnesium trisilicate (asbestos free), E 553b talc (asbestos free), E 554 sodium aluminum silicate and E 556 calcium aluminum silicate.
  • Flavor enhancers in the context of this invention are taken to mean naturally occurring or synthesized substances which are able to round off or enhance the flavor of foods. These also include flavorings. Examples which may be mentioned are: E 620 glutamic acid, E 621 monosodium glutamate, E 622 monopotassium glutamate, E 623 calcium diglutamate, E 624 monoammonium glutamate, E 625 magnesium diglutamate, E 626 guanylic acid, E 627 disodium guanylate, E 628 dipotassium guanylate, E 629 calcium guanylate, E 630 inosinic acid, E 631 disodium inosinate, E 632 dipotassium inosinate, E 633 dicalcium inosinate, E 634 calcium 5-ribonucleotide, E 635 disodium 5-ribonucleotide, E 640 glycine and E 650 zinc acetate.
  • the inventively used preparation can comprise aids.
  • Aids are taken according to the invention to mean substances which serve to improve the product properties, such as dusting behavior, flow properties, water absorption capacity and storage stability. Aids can be based on sugars, e.g. lactose or maltose dextrin, based on cereal or legume products, e.g. corn cob meal, wheat bran and soybean meal, based on mineral salts, inter alia salts of calcium, magnesium, sodium or potassium, and also D-pantothenic acid or its salts themselves (D-pantothenic acid salt produced chemically or by fermentation).
  • the inventively used preparations can comprise carriers.
  • Suitable carriers are "inert" carrier materials, that is to say materials which do not display adverse interactions with the components used in the inventive preparation. Obviously, the carrier material must be safe for the respective uses as aid, for example in foods and animal feedstuffs.
  • Suitable carrier materials are not only inorganic carriers but also organic carriers. Examples of suitable carrier materials which may be mentioned are: low-molecular-weight inorganic or organic compounds and also relatively high-molecular-weight organic compounds of natural or synthetic origin.
  • suitable low-molecular-weight inorganic carriers are salts, such as sodium chloride, calcium carbonate, sodium sulfate and magnesium sulfate, kieselguhr or silicic acid, or silicic acid derivatives, for example silicon dioxides, silicates or silica gels.
  • suitable organic carriers are, in particular, sugars, for example glucose, fructose, sucrose and also dextrins and starch products.
  • starch and cellulose preparations such as in particular corn starch, corn cob meal, ground rice hulls, wheat semolina bran or cereal flours, for example wheat, rye, barley and oat flour or brans and mixtures thereof.
  • the inventively used preparations can comprise the further constituents, carriers and aids in mixtures.
  • the weight fraction of the conjugated linoleic acid in the preparations can vary in wide ranges and is generally orientated according to practical considerations which result from the chosen field of application (for example farm animal husbandry, raising domestic animals or human nutrition).
  • the preparations are produced in the simplest case by mixing the constituents. Likewise, they can be produced by mixing solutions of the individual components, and if appropriate subsequently removing solvents.
  • the mixtures of various constituents can be present in any weight ratios to one another.
  • the simplest form of the mixture is bringing together the constituents in a mixer.
  • Such mixers are known to those skilled in the art, for example from the Ruberg company (vertical twin-shaft mixer (type HM (10-50 000 I)), ring-layer mixer-pelletizer (type RMG), continuous agglomerator dryer (type HMTK), vertical single-shaft mixer (type VM (10-50 000 I)), container mixer (type COM (50-4000 I)). Further mixers can also be obtained from Lodige, Drais, Engelsmann.
  • the mixers can be operated batchwise or continuously. In the batchwise mixer, generally all constituents to be mixed are charged in the desired ratio and then mixed for an adequate time in the region of minutes to hours.
  • the mixing time and the mixing stress are specified so that the constituents are present homogeneously distributed in the mixture.
  • the constituents are added continuously, if appropriate after premixing.
  • the residence time and mixing stress are to be chosen in such a manner that the constituents are present homogeneously distributed in the mixture.
  • the mixing time is frequently shorter in the continuous case and the stress is higher than in the case of batchwise mixing.
  • the mixing is customarily performed at room temperature, but can also, depending on the substances used, be carried out at higher or lower temperatures.
  • the preparations are present in solid form. Depending on the application requirement, the preparations can be powders having a mean particle size of from 10 ⁇ m to 5000 ⁇ m, preferably having a mean particle size of from 20 ⁇ m to 1000 ⁇ m .
  • the resultant particle size distribution of the pulverulent products can be studied in an instrument from Malvern Instruments GmbH, Mastersizer S.
  • the powders can be produced by crystallization, precipitation, drying, pelleting or agglomeration methods familiar to those skilled in the art, or other methods for forming solids described in current textbooks.
  • the exact amount of CLA to be incorporated into a dietetic food depends upon the intended use of the food, the form of CLA employed and the route of administration. It also can depend upon the isomer ratios. However, the dietetic food will contain the equivalent of about 0.05 to about 1 %, or about 0.1 % to about 0,9 %, or 0.2% to about 0.8%, or 0.3% to about 0.7%, or 0.4% to about 0.6% of CLA by weight of the dietetic food. In an additional embodiment the food will contain the equivalent of about 1 % to about 10 %, or 2% to about 8%, or 3% to about 7% , or 4% to about 6% of CLA by weight of the dietetic food.
  • the CLA content can also be expressed as the amount of CLA based on the total calories in the serving, e.g. 0.03 to 3 gram CLA per 100 calorie serving.
  • the amount of CLA can also be expressed as a percentage of the lipid or fat in the food, such as 0.3% to 100% of the food lipid.
  • European patent application EP779033 A1 discloses an edible fat spread containing 0.05 to 20% (by weight) CLA residues.
  • a commercially-available mixture of free fatty acids having a linoleic acid content of 95,3% was subjected to alkali isomerization with NaOH in ethylene glycol.
  • the free fatty acids were incorporated into triglycerides by mixing with 10 parts palm oil and lipase. The mixture was stirred for 48 hours at 45°C and the lipase and free fatty acids removed. Seventy parts of this compositions and 29 parts water.
  • This application also discloses a baby formula containing similar amounts of CLA along with 2.66 gm of protein, 5.46 gm of fat, 10.1gm of carbohydrate, 133 gm of water, and vitamins and minerals in RDA (Recommended Daily Allowance) amounts.
  • Another example of a low-residue liquid enteral dietetic product useful as a high-protein, vitamin and mineral supplement is disclosed. This supplement contains CLA at 0.05% to about 5% by weight of the product, or by 0.3% to about 100% of the lipid present or about 0.03 to 0.3 gm CLA per 100 calories.
  • 140 calories of a representative formula can contain 7.5 gm of egg white solids, 0.1 gm CLA, 27.3 gm carbohydrate such as sucrose or hydrolyzed cornstarch, 1.9 gm of water, and vitamins and minerals in RDA amounts.
  • the invention relates to transgenic microorganism expressing a nucleic acid molecule as described above encoding a trans-10, cis-12 conjugated linoleic acid isomerase characterized by a sequence
  • nucleic acid molecule having the sequence as described in SEQ ID No. 1 or (ii) from functional equivalents of the polypeptide encoded by the nucleic acid molecule described in (i) such as: e. a nucleic acid molecule having at least 50, preferably at least 75, more preferably at least 100, especially preferably at least 125, very especially preferably at least 150 consecutive base pairs of the sequence described by SEQ ID No.1 , or f. a nucleic acid molecule having an identity of at least 80%, preferably at least
  • nucleic acid molecule encoding a polypeptide having at least 75%, preferably at least 85%, more preferably at least 90%, especially preferably at least 95%, very especially preferably at least 98% identity to the amino acid sequence as shown in SEQ ID No. 2.
  • said nucleic acid sequence is preferably isolated from a rumen bacteria, more preferably from Megashera elsdenii, most preferably from Megashera elsdenii YJ-4, or from a microorganism belonging to the genus Propionibacterium, preferably Propionibacterium acnes , wherein said nucleic acid molecule is functionally linked to at least one heterologous promoter sequence.
  • the present invention relates to the use of the inventive transgenic microorganism, preferably microorganism belonging to the genus selected from the group consisting of Lactococcus, Lactobacillus, Propionibacterium, Escherichia and Bifidobacterium, as desribed above, more preferably microorganism selected from the group consisting of Bifidobacterium breve, Bifidobacterium dentium and Bifidobacterium pseudocatenulatum as probiotics in food and feed.
  • the inventive transgenic microorganism preferably microorganism belonging to the genus selected from the group consisting of Lactococcus, Lactobacillus, Propionibacterium, Escherichia and Bifidobacterium, as desribed above, more preferably microorganism selected from the group consisting of Bifidobacterium breve, Bifidobacterium dentium and Bifidobacterium pseudocatenulatum as probiotic
  • the invention relates to fermented oil produced in transgenic microorganism according to the above described inventive process.
  • the fermentative oil is isolated from the fermentation broth and consist mainly of trans-10, cis-12 octadecadienoic acid and 9,12-Octadecadienoic acid and is enriched in trans-10, cis-12 octadecadienoic acid to at least 20%, 30%, 40%, preferably at least 50%, 55%, 60% more preferably at least 65%, 70%, 75% especially preferably at least 80%, 85%, 90% very especially preferably at least 91 %, 92%, 93%, 94%, 95%.
  • said fermented oil can be further processed (see example).
  • the invention relates furthermore to the use of the fermented oil produced according to the above described inventive method for
  • the amount generally ranges from about 0.001 g/kg to about 1g/kg of the animal body weight.
  • chicks fed a 0,5% CLA diet and subsequently challenged with a wing web injection of live attenuated fowl pox virus gained more weight than chicks fed a control diet.
  • Chicks fed the 0.5% CLA diet demonstrated a markedly enhanced percent of CD-4 and CD-8 cells as compared to chicks fed a control diet.
  • CLA isomers have previously been reported to decrease cell viability and stimulate apoptosis in SW480 cells.
  • the 10, c12 CLA isomer was the most potent isomer, which reduced cell viability by 47-61 % compared with 40-52 % reduction by the c9, t11 CLA.
  • CLA has also been shown to inhibit growth of the MCF-7 breast cancer cell line (Schultz et al., 1992; O'Shea et al., 1999).
  • MCF-7 cells were treated with 20 ⁇ g/ml of the t10, c12 CLA isomer for 8 days, a 15 % decrease in cell numbers was observed, whereas the same amount of the c9, t11 CLA isomer caused a 60 % decrease in cell viability during same conditions (O'Shea et al., 1999).
  • the t10, c12 CLA isomer reduced viability by 50-60 % in both SW480 and MCF-7 cell lines following 4 days incubation with 16 ⁇ g/ml (Miller et al.,
  • Medicaments and therapeutic agents are taken to mean those agents which are used not only for prevention, but also for therapeutic treatment of allergic reaction, increased body fat, anorexia and weight loss due to immune stimulation, blood lipid profiles and cancer in animals, preferably in human.
  • the preparations can be formulated in a manner which is generally known to those skilled in the art and is suitable and can be used for the production of pharmaceutical dosage forms with the use of conventional techniques. Such techniques are described, for example, in "Remington ' s Pharmaceutical Science Handbook", Mack Publishing Co., New York, USA, 17 th edition 1985.
  • Such pharmaceutical dosage forms or food additives can be liquids, powders, premixes, tablets, capsules or suspensions.
  • compositions will generally range from about 1 ,000 parts per million (ppm) to about 10,000 ppm of CLA of the human ' s diet. However, the upper limit of the amount to be employed is not critical because CLA is nontoxic. CLA for this and other uses may also be prepared in a variety of forms. These include nontoxic sodium or potassium salts of CLA in combination with pharmaceutical diluent and active esters. CLA may also be incorporated directly into animal feed or food to be fed to a human so that CLA comprises approximately 0.01 % to 2% or more by weight of the animal or human ' s food.
  • Lactococcus lactis NZ9800 (a L. lactis NZ9700 derivative which does not produce nisin because of a deletion in the nisA gene, and contains the nisRK signal transduction genes integrated on the chromosome) was cultured at 30 0 C in M17 (Difco laboratories, Detroit Michigan, USA) broth and/or agar containing glucose (0.5% w/v).
  • the probiotic strain Lactobacillus paracasei ssp. paracasei NFBC 338 (Lb. paracasei NFBC 338) was previously isolated from the human gastrointestinal tract (GIT), and obtained from University College Cork, Ireland under a restricted materials transfer agreement. Lb.
  • paracasei NFBC 338 was routinely cultured overnight ( ⁇ 17 h) in MRS broth (Oxoid Ltd., Hampshire, UK) and incubated at 37°C under anaerobic conditions using anaerobic jars containing Anaerocult A gas packs (Merck, Darmstedt, Germany).
  • L. lactis carrying the plasmids pNZ44 were routinely cultured in the presence of chloramphenicol (5 ⁇ g/ml) as a selective marker.
  • Lb. paracasei NFBC harboring the vector pMSP3535 were routinely cultured with erythromycin (10 ⁇ g/ml) as selective marker.
  • coli TOP 10 (Invitrogen) harbouring the plasmid pNZ44 was cultured in LB (Luria-Bertani)- media supplemented with chloramphenicol (20 ⁇ g/ml).
  • Human colon cancer cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).
  • the t10, c12 CLA isomer (98 %+ purity) was obtained from Matreya (Matreya Inc., PA, USA;).
  • Cell culture media and supplements were purchased from Sigma Aldrich Ireland Ltd. (Dublin, Ireland), unless otherwise stated.
  • SW480 cells were maintained in Dulbecco's Minimum Essential Medium (DMEM) supplemented with 5% (v/v) fetal bovine serum, 0.2 mM L- glutamine, 1 mM HEPES and 1 unit/ml penicillin and streptomycin.
  • DMEM Dulbecco's Minimum Essential Medium
  • SW480 cells were grown in 96 well plates and maintained at 37 0 C in a humidified atmosphere and a pH of 12-1 A by a required flow of 95 % air and 5 % CO2.
  • Example 2 DNA manipulation. Two oligonucleotide primers were designed to amplify the complete linoleic acid isomerase (coPAI) for production of t10, c12 CLA from the original construct pC33.1-coPAI (linoleic acid isomerase gene in a plant vector; BASF, Germany).
  • the forward primer designated ERcoPAM (SEQ ID No. 3) contains a Pstl restriction site and a ribosome binding site (RBS), four extra bases at the 5'end and seven extra bases between the RBS and the gene start; 5'- AAAACTGCAGAGGAGGAAAAAAAAAATGGGTTCCATTTCCAAGGA - 3' (SEQ ID No. 3).
  • the reverse primer designated ERcoPAI2 (SEQ ID No. 4)contains a Kpnl restriction site and three extra bases at the 5' end; 5'- CGGGGTACCTCACACGAAGAACCGCGTCA - 3' (SEQ ID No.: 4).
  • the 1278 bp coPAI gene was amplified in an Eppendorf Mastercycler Gradient (Eppendorf) with High Fidelity Expand as described by the supplier (Roche Diagnostics Limited, East Wales, England) using 200 ng plasmid DNA (PC33.1-COPAI) as a template.
  • PCR reactions were performed in a total volume of 50 ⁇ l containing 1 ⁇ l of each primer, 3 mM MgCb, 5 ⁇ l 10x Expand buffer, 1 ⁇ l dNTP's and 0.75 ⁇ l Expand DNA.
  • PCR conditions were as follows; 10 cycles of 2 min, 15 s denaturation (94°C), 30 s annealing (55°C), 2 min elongation (72°C) followed by 20 cycles of 15 s (94°C), 30 s (55°C), 2 min + 5 s/cycle (72°C) and finally, one 7 min cycle at 72°C.
  • the PCR reaction mixture was analysed on a 1 % (w/v) agarose gel to visualize the resulting PCR fragment.
  • the Qiagen Plasmid Mini kit (Qiagen, West Wales, UK) was used to isolate plasmid DNA from E. coli TOP 10, L. lactis NZ9800, and Lb. paracasei NFBC 338 with one minor modification for L. lactis and Lb. paracasei, i.e. 40 mg/ml lysozyme was added to buffer P1 and incubated for 20 min (L. lactis) and 2 hours (Lb. paracasei) at 37°C. PCR products were purified using a Qiaquick PCR Purification Kit (Qiagen).
  • the two plasmids pNZ8048 (Nisin inducible plasmid containing PnisA promoter) and pNZ44 (a derivative of pNZ8048 in which the PnisA promoter is replaced by P44, a constitutive L. lactis chromosomal promoter) and the coPAI gene fragment were restricted with Pstl and Kpnl followed by ligation reaction at 15°C with T4 DNA ligase as described by the supplier (New England Biolabs, MA USA (NEB). The construct is shown in Fig. 1. Recombinant plasmids were double digested with the same enzymes to verify the correct clone and then electroporated into L. lactis NZ9800.
  • the gene was cut out of pNZ8048-coPAI using Pstl and Xbal restriction enzymes (Fig. 1) and ligated into the same sites of the Lactobacillus nisin inducible vector pMSP3535.
  • Electrocompetent L. lactis were prepared and transformed according to the method described by de Ruyter et al., while electrocompetent Lb. paracasei NFBC 338 cells were prepared using 3.5X SMEB (1 M sucrose, 3.5mM MgCI ⁇ ) as described by Luchansky et al.. Sequence analysis was performed using DNAStar software (DNAStar, Madison, Wisconsin, USA).
  • Example 3 Screening for CLA production.
  • Cis-9, trans-11 and trans-10, cis-12 CLA standards were purchased from Matreya (Matreya Inc., PA, USA) and linoleic acid from Sigma (Sigma Chemical, MO, USA).
  • the L. lactis, Lb. paracasei and E. coli clones were tested for their ability to convert free linoleic acid (0.1-0.5 mg ml i ) to trans-10, cis-12 CLA as follows; 1 % inoculum of an overnight culture was transferred to 10 ml broth and incubated until the culture reached OD6oonm ⁇ 0.5.
  • linoleic acid (0.1-0.5 mg/ml) was added to cultures and inducible cultures were induced with 30-50 ng/ml nisin (prepared from milk solids containing 2.5% (w/v) nisin, Sigma, N-5764) followed by further incubation for 48-72 h.
  • Cultures subjected to time experiments were grown in a larger volume of broth and 10 ml samples were taken every 12 h.
  • the culture was centrifuged and fatty acids were extracted from the supernatant and dried down under a nitrogen stream followed by methylation and analysis by gas liquid chromatography (GLC) as described (Coakley et al, 2003). All conversion rates in percentage are related to the amount of linoleic acid that was recovered and extracted from the media following incubation without culture for the same time as with culture, which represented 100 % of available linoleic acid.
  • GLC gas liquid chromatography
  • a linoleic acid control consisting of uninoculated media containing linoleic acid (0.5 mg/ml) was also prepared and incubated at 37 0 C for 72 hours, followed by extraction of the fatty acids.
  • Control samples prepared in triplicate from each fermentations and the unfermented linoleic acid control were also methylated and analyzed on GLC as described (Coakley et al., 2003) to calculate the ratio CLA/linoleic acid present in the sample.
  • Example 5 Anti-proliferative activity of fermented oils on human SW480 colon cancer cells.
  • the oils extracted following fermentation of the cultures L. lactis pNZ44-coPAI and E. coli pNZ44-coPAI
  • respective media containing linoleic acid 0.5 mg/ml
  • the 1278 bp gene (accession no CQ766028) from Propionibacterium acnes encodes a linoleic acid isomerase protein for t10, c12 production of 425 amino acids (SEQ ID No. 1).
  • the molecular weight of the isomerase is 49,077 Da.
  • Comparison with sequences in the database revealed that the cloned isomerase protein showed significant homology with proteins known as amino oxidases over most of the sequence ( ⁇ a.a 25 - 400; NCBI conserveed Domain Search, Marchler-Bauer et al., 2005).
  • the isomerase showed 96 % identity to a putative amino oxidase from Propionibacterium acnes (accession no Q6A8X5_PROAC; EXPASY/UniProtKB database), but only 26 % identity to the next best match, a protein from the plant Oryza sativa (japonica cultivar-group, accession no Q7XR12_ORYSA; EXPASY/UniProtKB database) spanning from amino acid 145-423.
  • the aligned region includes a flavin-binding site in these proteins.
  • the flavin containing amine oxidase family also contains phytoene hydrogenases and related enzymes.
  • An NAD/FAD binding domain located in the region between amino acid residue 10- 39 was identified (PROSITE database).
  • the isomerase protein is soluble and the predicted location of the protein is cytoplasmic (PSORTb, British Columbia, Canada; SOSUI, Mitaku
  • Example 7 Bioconversion of linoleic acid.
  • L. lactis carrying the construct pNZ44-coPAI was shown to convert free linoleic acid into t10, c12 CLA, compared with control culture L. lactis containing only the vector pNZ44, with which no conversion to CLA was detected (Table 1). L. lactis pNZ44-coPAI converted as much as
  • paracasei NFBC 338 harboring the lactobacilli vector and the coPAI gene, pMSP3535-coPAI, converted nearly 30 % of the LA (recovered in a control media after incubation without culture) following induction at OD6oo 0.5 with 50 ng/ml nisin and incubation for 48 hours in the fatty acid (0.5 mg/ml).
  • OD6oo 0.5 with 50 ng/ml nisin and incubation for 48 hours in the fatty acid (0.5 mg/ml).
  • Example 8 Isolation of lipids from the microorgansims
  • the Bifidobacterium strain was grown (2 % inoculum) in 500 ml cys-MRS (0.05 % (w/v) L- cysteine hydrochloride (98 % pure; Sigma Chemical Co. St. Louis, MO, USA) was added to the MRS medium) with 0.5 mg ml 1 added linoleic acid (Sigma Chemical Co.) to assess bioconversion of the substrate.
  • the linoleic acid was added as a 30 mg ml 1 stock solution in distilled water containing 2 % (v/v) Tween 80.
  • the linoleic acid stock solution was previously filter-sterilised through a 0.45 mm Minisart filter and stored in the dark at -20 0 C.
  • the strains were incubated anaerobically for 42 hours at 37 0 C. Following incubation, the fatty acids in the bacterial supernatant was extracted as follows: to 450 ml of the bacterial supernatant, 225 ml isopropanol (99 % purity; Alkem Chemicals Ltd., Cork, Ireland) was added and vortexed for 30 sec. Hexane (170 ml added initially and vortex mixed before adding a further 340 ml hexane) (99 % purity; LabScan Ltd., Dublin, Ireland) was added to this mixture, vortexed and centrifuged at 960 x g for 5 min.
  • the resultant supernatant (hexane layer containing lipids) was removed to a glass tube and the hexane was dried to 2-3 ml under a stream of nitrogen at 45°C.
  • the lipids were stored under nitrogen at -2O 0 C.
  • Fatty acid compostion of the bacterial supernatant and level of conversion of the linoleic acid to CLA was assessed following addition of an internal standard (C130 tridecanoic acid (99 % pure, Sigma Chemical Co.), methylation and gas liquid chromatography (GLC), as previously described (Stanton et al., 1997).
  • Example 9 Preparation of Fatty Acid Methyl Esters (FAME) and GLC Analysis
  • FAME Fatty Acid Methyl Esters
  • GLC GLC following acid-catalyzed methylation as described previously (Stanton et al., 1997).
  • Free fatty acids in oils such as sunflower and soybean oils were calculated as the difference between fatty acid concentrations obtained following acid and base catalyzed methylation, performed using 2 N methanolic KOH (Sigma Chemical Co.) at room temperature.
  • the GLC was performed with reference to the internal standard C130. Separation of the FAME was performed on a Chrompack CP SiI 88 column (Chrompack, Middleburg, The Netherlands, 100 m x 0.25 mm i.d., 0.20 ⁇ m film thickness), using helium as carrier gas at a pressure of 37 psi.
  • the injector temperature was held isothermally at 225°C for 10 min and the detector temperature was 250_C.
  • the column oven was held at an initial temperature of 140 0 C for 8 min and then programmed at an increase of 8.5°C/min to a final temperature of 200 0 C, which was held for 41 min. Collected data were recorded and analyzed on a Minichrom PC system (VG Data System, Manchester, UK). The trans-10, cis-12 CLA isomer CLA isomer was identified by retention time with reference to a CLA mix (Nu-Chek- Prep. Inc., Elysian, MN).
  • the percentage conversion to CLA and the remaining linoleic acid in the broth were calculated by dividing the amount of CLA and linoleic acid present in the broth after inoculation and incubation with the various cultures used with the amount of linoleic acid present in the spiked broth before incubation.
  • Example 10 Lipid extraction of supernatant After transferring 10 ml of the cultures inoculated with either CLA or LA to 15 ml centrifuge tubes (Sarstedt, Numbrecht, Germany), centrifugation was performed at 2197 x g for 20 min at room temperature (20 0 C), using a Sanyo Mistral 2000 R centrifuge. To 4 ml of the supernatant were added 0.75 mg C 13:0 (tridecenoic acid, Sigma, 99% pure) as internal standard prior to lipid extraction, performed as follows: 2 ml isopropanol (Alkem Chemicals Ltd. Cork, Ireland, 99% purity) and 1.5 ml hexane (LabScan Ltd.
  • Example 11 Lipid extraction of pellet
  • bacterial cells from 10 ml of grown culture were washed by adding and resuspending them in 1 ml saline solution (0.137 M NaCI, 7.0 mM K2HPO4,
  • Acid catalyzed methylation which results in derivatisation of both free fatty acids and triglyceride bound fatty acids was performed as described below: Extracted lipids from supernatants and pellets (as described in sections 2.4.1 and 2.4.2) in screw capped glass tube, were resuspended in 12 ml, 4% methanolic HCI (v/v) (Supelco Inc. Bellefonte, PA, USA) in methanol and vortex mixed for 10 sec. The lipids in methanolic HCI were incubated at 6O 0 C for 1 h with vortex mixing every 10 min.
  • the free fatty acids were analysed as fatty acid methyl esters (FAME) using a gas liquid chromatograph (GLC-Varian 3400, Varian, Harbor City, CA, USA) fitted with a flame ionization detector (FID) and a Septun Programmable Injector (SPI). Quantification of fatty acids was performed with reference to the internal standard (C 13:0). Separation of fatty acids was performed on a Chrompack CP SiI 88 column (Chrompack, Middleburg, The Netherlands) (100 m x 0.25 mm i.d., 0.20 m film thickness), using He as carrier gas at a pressure of 33 psi.
  • FAME fatty acid methyl esters
  • the injector temperature was held isothermally at 225 0 C for 10 min and the detector temperature was 25O 0 C.
  • the column oven was held at an initial temperature of 14O 0 C for 8 min, and then programmed at an increase of 8.5C/min to a final temperature of 200 0 C, which was held for 41 min.
  • trans-10, cis-12 CLA isomer was identified by retention time with reference to CLA standards (Matreya Inc. PA, USA), and trans-11-C 18:1 and stearic acid (Sigma Chemical Co. St. Louis, MO, USA) identified by reference to their standard fatty acids.
  • Cfi (Ais x Wti) / (Ai x Wtis), where Cfi is the correction factor for the actual CLA isomer, Ais is refers to the area of the internal standard (C 13:0), Ai is the area of the CLA peak, Wti is the weight of the CLA isomer and Whs refers to the weight of the internal standard.
  • the quantity of CLA was expressed as mg/ml broth.
  • the response factors of the individual fatty acids were calculated relative to the area of C 18:0, which was assigned a response factor of 1.00.
  • the % conversion to CLA and the % remaining linoleic acid in the broth were calculated by dividing the amount of CLA and linoleic acid present in the broth after inoculation with the cultures used, with the amount of linoleic acid present in the spiked broth before incubation. All conversion rates in percentage are related to the amount of linoleic acid that was recovered and extracted from the media following incubation without culture for the same time as with culture, which represented 100 % of available linoleic acid
  • Example 14 Anti-proliferative activity of fermented oils on human colon cancer cells SW480.
  • human colon cancer cells SW480 were cultured in the presence of the extracted fermented oils consisting of a mixture of linoleic acid and t10, c12 CLA at a ratio of ⁇ 1.35:1.
  • Controls of linoleic acid extracted from LB broth after 72 hours incubation at 37 0 C, linoleic acid (Sigma, 95%) and the pure synthetic t10, c12 CLA isomer (Matreya) were also cultured with SW480 cells to compare the effect of the fermented oils versus the pure isomer, but also to ensure that the concentration of the added oils were below the concentration when linoleic acid starts to have a cytotoxic effect on the cancer cells.
  • linoleic acid has been shown to have an anti-proliferative effect on SW480 cancer cells at 42.8 ⁇ g/ml media (152.5 ⁇ M), and a slightly proliferative effect at a concentration of 16.9 ⁇ g/ml media (60.2 ⁇ M) (Miller et al., 2003), concentrations of t10, c12 CLA (fermented oil samples) between 5-20 ⁇ g/ml media (equivalent to 6.7-27 ⁇ g of linoleic acid/ml media in the same oil sample) were chosen so as not to exceed the threshold concentration when linoleic acid inhibits cell growth.
  • Controlled gene expression systems for lactic acid bacteria Transferable nisin-inducible expression cassettes for Lactococcus, Leuconostoc, and Lactobacillus spp. Appl. Environ. Microbiol. 63: 4581-4584.
  • CLA Conjugated linoleic acid

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Abstract

La présente invention concerne un procédé, destiné à produire un acide linoléique conjugué trans-10, cis-12 dans un microorganisme transgénique, qui comprend les étapes consistant à : (a) introduire dans ledit microorganisme au moins une molécule d’acide nucléique codant pour l’isomérase de l’acide linoléique trans-10, cis-12 ; (b) cultiver le microorganisme transgénique obtenu au cours de l’étape (a) ; (c) induire la production de l’acide linoléique conjugué trans-10, cis-12 en ajoutant un acide linoléique dans la culture ; (d) incuber ladite culture pendant au moins 12 heures ; et (e) isoler l’acide linoléique conjugué du milieu de culture et/ou du microorganisme transgénique.
PCT/EP2006/069030 2005-12-24 2006-11-29 Procede de production de l’acide trans-10, cis 12 octadecadienoique Ceased WO2007074010A1 (fr)

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JP2008546335A JP2009521213A (ja) 2005-12-24 2006-11-29 トランス−１０，シス−１２オクタデカジエン酸を製造する方法

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